Topics in Bull Fertility

Chenoweth P.J. (Ed.) Large Animal Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

Index of content

1. Bull Breeding Soundness Evaluation: Current Status (Page 2) 2. Evaluation of Frozen Semen: Traditional and New Approaches (Page 8) 3. Sexing Bull Sperm (Page 13) 4. Scrotal/Testicular Thermoregulation in Bulls (Page 16) 5. Management and Economics of Natural Service Sires on Dairy Herds (Page 20) 6. Management and Evaluation Considerations for Range Beef Bulls (Page 25) 7. Bull Sex Drive and Reproductive Behavior (Page 27)

Bull Breeding Soundness Evaluation: Current Status

J. C. Spitzer

Department of Animal and Veterinary Sciences, Clemson University, Clemson, South Carolina, USA.

Bull Breeding Soundness Evaluation A breeding soundness evaluation (BSE) is a relatively quick and economically prudent procedure for screening

bulls in relation to potential fertility. Conducting a BSE implies a complete evaluation of all factors contributing to

normal reproductive potential. Veterinarians are advised to be as thorough as current technologies allow and to

consistently follow a routinely standardized procedure. Predicting which bulls would be least successful in

impregnating cows represents the greatest value of the current system. That is, based on history and information

collected at evaluation, bulls are categorized as being above or below thresholds for characteristics known to

most affect fertility. The most recent guidelines for BSE were approved by the Society for Theriogenology (SFT) in

1992 [1,2]. The SFT also makes Bull Breeding Soundness Evaluation forms (individual bulls) and Bull Certificate

of Evaluation forms (herds) available to members (SFT, 530 Church Street, Suite 700, Nashville, TN 37219:

therio@wmgt.org).

As outlined, a BSE is intended to be a rapid, albeit thorough and systematic format for identifying problems

affecting male fertility. A BSE consists of three steps, as follows;

1. A generalized physical examination and thorough examination of both internal and external portions of

the reproductive system;

2. A scrotal circumference measurement; and

3. Collection and evaluation of a semen sample. The SFT format for bulls established minimum acceptable

thresholds for scrotal circumference, sperm motility and sperm morphology (Table 1).

Table 1. Reference tables for evaluation of scrotal circumference and spermiogram. (Reprinted with

permission of the Society for Theriogenology. BSE form is copyrighted by the Society, it is for use of

members only, and cannot be duplicated).

Minimum Recommended Motility is: 30% or FAIR (F)

Mass Activity (Gross) Rating Individual

Rapid Swirling Very Good

(VG) ≤ 70%

Slower Swirling Good (G) 50 - 69%

Generalized Oscillation

Fair (F) 30 - 49%

Sporadic Oscillation Poor (P) < 30%

To be classified a satisfactory potential breeder, requires a satisfactory physical examination and minimum

values for scrotal circumference, motility and morphology. Any bull not meeting minimums is classified either as

an unsatisfactory potential breeder or classification may be deferred at the discretion of the evaluator. Interpretation of Results Satisfactory Potential Breeder Bulls achieving the minimum threshold values for scrotal circumference, sperm motility and sperm morphology.

These bulls must also be free of genetic, skeletal, infectious or other problems or injuries which might

compromise fertility in natural mating situations. Unsatisfactory Potential Breeder Bulls which fail to meet minimum threshold values in any category and which have a poor prognosis for

improvement. Any bull having a significant physical defect which would interfere with successfully impregnating

cows should be classified as unsatisfactory. Any bull having a significant problem involving the reproductive

system which is unlikely to improve should also be classified as unsatisfactory, as well as any bull of less than the

minimum scrotal circumference (per age category). Any bull providing an ejaculate containing sperm less than

30% motile or less than 70% normal morphology should be classified as either unsatisfactory or deferred. Classification Deferred Bulls which cannot currently be rated satisfactory but are likely to improve with time or therapy. Provision is made

on the SFT form for scheduling of a retest. This category includes young bulls with immature semen profiles as

well as any bull which has poor semen quality considered to be capable of improvement. Also in this category are

bulls failing to yield an acceptable ejaculate for reasons which are not obvious despite careful physical

examination, as well as bulls with problems which are considered to be treatable. If there is doubt that a bull may

be confidently assigned to either the satisfactory or unsatisfactory potential breeder category, a retest is advised

and the bull should be assigned to the classification deferred category. Both false positives and false negatives

are unacceptable as it is equally bad to condemn a satisfactory bull as to approve one that should be classified as

unsatisfactory. Rational for Significance of Breeding Soundness Evaluation As recently reviewed by Chenoweth, the reproductive capabilities of breeding bulls are essential to any

consideration of cow/calf economics [4]. In the 1994 NAHMS survey [5] physical unsoundness and infertility were

identified as the most important culling criteria for beef bulls. Clients must be convinced of the real economic

significance of identifying unsound and/or subfertile bulls prior to purchase and/or use.

While few bulls are completely sterile, even subfertile bulls adversely affect pregnancy rates. One of the earliest

studies (using BSE standards in effect at that time) employed 48 bulls classified as satisfactory, questionable, or

unsatisfactory potential breeders with natural mating achieving pregnancy rates of 75, 52, and 12%, respectively

[6]. While fertility prediction for individual bulls was imperfect, use of bulls which failed to meet satisfactory

potential breeder criteria resulted in considerably less pregnant females than did use of satisfactory bulls.

Two studies conducted by Wiltbank and Parish are germane to any discussion of the relationship between

economics and the BSE [7]. Both trials utilized large multi-sire herds with cow to bull ratios of approximately 25:1.

In these studies, all available bulls were subject to a BSE and bulls failing to pass the physical examination or

having inadequate scrotal circumference were eliminated. In trial 1, cows were either mated with bulls of at least

80% normal sperm morphology, or with a group of control bulls. In trial 2, virgin heifers were mated with bulls of

either at least 70% or 80% normal sperm morphology, or with a group of control bulls. In both trials, the control

bulls had the array of percent normal sperm found in the original, larger population of available bulls.

In trial 1, using bulls with at least 80% normal sperm resulted in a 93% pregnancy rate compared with an 87%

pregnancy rate for control bulls. In trial 2, pregnancy rates were 90% for heifers mated to bulls with at least 80%

normal sperm, 91% for heifers mated to bulls with at least 70% normal sperm and 85% for heifers mated to

control bulls. On average, bulls with acceptable physical traits and meeting minimum scrotal circumference

guidelines and preselected to have at least 70% normal sperm resulted in 5 to 6% more cows pregnant than bulls

unselected for normal sperm. Such differences could be expected to be even greater in single sire herds.

In another study, the most important factors influencing pregnancy rates were large scrotal circumference and

relative lack of primary sperm defects [8]. These studies as well as others support the 1993 SFT guidelines which

place increased emphasis on a minimum scrotal circumference based on bull age, as well as a minimum

acceptable threshold of 70% normal sperm. Outcomes of Breeding Soundness Evaluation A recent analysis of BSE for 2898 yearling (10 to 20 months) beef bulls indicated that 72% were satisfactory

potential breeders [9]. These data were from five University sponsored bull testing programs in 2 states, with the

percentage of bulls classified as satisfactory varying from 65 to 79% among locations. Apparently there was

considerable genetic variation in reproductive capabilities among bulls consigned to various test stations.

However, in general, 28% of these young bulls did not achieve satisfactory potential breeder status within the time

constraints of these testing programs. Interestingly, of the 28% of bulls classified as unsatisfactory potential

breeder; 4% failed to pass the physical examination, 9% were below minimum scrotal circumference based on

age, 9% had less than 70% normal sperm, 4% had less than 30% progressive motility, and 4% had both less than

70% normal sperm and less than 30% progressive motility (numbers do not sum due to rounding). Age affected

the classification outcome (Fig. 1) as 10 month-old bulls were less likely to be classified as satisfactory than bulls

greater than 11 months of age. There was also a tendency for fewer 11 month-old bulls to be classified as

satisfactory than bulls greater than 12 months of age.

Figure 2. Overall percentage of bull classified as satisfactory potential breeders among breeds of bulls evaluated. Bars with different superscripts differ (P<0.05).

A retrospective analysis of BSE as classified by the 1993 SFT guidelines [1] on 3648 bulls of 10 breeds has

recently been completed [10]. Of these bulls, 76% were classified as satisfactory potential breeders. Both breed

(Fig. 2) and age (Fig. 3) were significant sources of variation for classification outcome.

Figure 3. Overall percentages of bulls classified as satisfactory potential breeders among ages of bulls evaluated. Bars with different superscripts differ (P<0.05).

Figure 4. Overall percentages of bulls classified as satisfactory potential breeders among ages of bulls evaluated. Bars with different superscripts differ (P<0.05).

Bulls classified as unsatisfactory potential breeders were most likely to be classified as such due to inadequate

normal sperm morphology or inadequate scrotal circumference. Most bulls receiving unsatisfactory classifications

due to inadequate scrotal circumference were 10 and 11-month-old bulls which may have been evaluated

prematurely. Santa Gertrudis bulls were the breed most likely to classify unsatisfactory due to inadequate scrotal

circumference as well as a combination of spermatozoal parameters. The proportion of bulls failing due to

abnormal sperm morphology was influenced most by breed; here Simbrah and Brangus bulls classified as

unsatisfactory potential breeders more often than bulls of the other breeds represented. Interpretation of these results is difficult due to inconsistencies across testing stations. Bos indicus influenced composite breeds such as

Santa Gertrudis mature more slowly than British or Continental breeds. This increases the chances that yearling

bulls of such breeds might be relatively deficient in scrotal circumference and spermatozoal parameters. However, bulls of Bos indicus derivation at some stations were able to classify as satisfactory potential breeders at rates

comparable with the other, non zebu breeds. It is possible that inconsistencies observed might be due to inadequate selection pressure for reproductive traits among some Bos indicus influenced populations, and that

such breed-types may achieve satisfactory BSE classification at rates equivalent to those in bulls of the

Continental and British breeds if they are exposed to adequate selection pressure for reproductive traits.

These reports confirm that the BSE performed on yearling performance-tested bulls, despite enormous variations

in ages, breeds, and populations of bulls is feasible, with approximately 70 to 80% of all bulls being classified as

satisfactory potential breeders.

Evaluation of Frozen Semen: Traditional and New Approaches

H. Rodriguez-Martinez

Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural

Sciences (SLU), Uppsala, Sweden.

Summary Subjective evaluation of sperm motility post-thaw is the single mostly used parameter to determine the quality of

bull semen intended for AI. Albeit being a good indicator for sperm viability and of relevance for fertility, post-thaw

motility is not a good predictor of the fertility level of the AI-semen dose. Such levels are determined by the AI of

several hundreds or thousands of females. This costly procedure has encouraged world-wide research looking for

alternative, or at least complementary, in vitro assessment methods. Such methods are hereby reviewed,

particularly those evaluating multiple functions of frozen-thawed bull AI semen, and their relationship with AI-

fertility. The combined outcome of a set of sperm functional parameters post-thaw; such as sperm linear motility,

total concentration and concentration of motile spermatozoa after swim-up, the frequency of uncapacitated

spermatozoa and their readiness to acrosome react when exposed to ionophores, their degree of chromatin

stability as well as their ability to bind homologous zona pellucida (zona pellucida binding assay, ZBA) and to

fertilize in vitro (IVF) has been significantly related (retrospectively) to the observed fertility of sires after AI.

Furthermore, a predicted fertility value has been obtained by in vitro evaluation of frozen-thawed semen,

indicating it is possible to eliminate sub-fertile bulls before their semen enter an AI program. Artificial Insemination (AI): The Most Successful Reproductive Biotechnology in cattle AI plays a dual role in cattle breeding. Firstly, it prevents spreading of venereal diseases and, secondly, it

effectively disseminates desirable genetic material from proven sires onto a female population, thus improving

their health status and the genetic gain of major populations (>150 million cows inseminated annually worldwide

with frozen-thawed semen). To warrant the highest possible outcome of AI, those sires genetically selected for

breeding are monitored andrologically for soundness and their semen is periodically examined for normality and

processed using the best known handling procedures including freezing and thawing. Ultimately, the processed

semen is deposited by AI taking care that sperm deposition is done as close as possible to expected ovulation.

Besides all these efforts the fertility after first AI is considered acceptable when the non-return to oestrus rate after

56 days of AI (56d-NRR) is ≥60%. Although many factors can account for AI-fertility, the fertilizing ability of the

frozen-thawed semen is, probably, the most important. Fertilizing Ability is Affected by Freezing-Thawing Spermatozoa are terminal cells, whose major role is to carry a genome/centriole package to the oocyte. For this

task, spermatozoa are equipped with a setup of specialized structures (a domain-marked plasma membrane,

diverse organelles such as mitochondria, a flagellum, an acrosome) that ensures a particular interaction with the

female genital tract and the oocyte. Their cellular viability decreases substantially within a short time after

ejaculation. Cryopreservation, which attempts to ensure their survival, imposes however, irreversible damage to

the sperm membranes that causes either cell death [1] or capacitation-like changes in the plasmalemma [2],

hampering their ability to fertilize. In order to maximize the number of AI´s that can be performed with a single

ejaculate from a proven sire, the AI industry has steadily decreased the number of spermatozoa per frozen semen

AI-dose and figures of 7.5-10 million are nowadays common [3]. With freezing-thawing affecting a large proportion

of the spermatozoa there is, for each individual sire, a threshold number of viable spermatozoa per AI-dose which,

ultimately, expresses a certain fertility level. Below this threshold sperm number, the fewer the viable

spermatozoa inseminated the greater the risk fertility drops [4]. A significant relation has been shown between

sperm number and achieved AI-fertility, which follows linearly the fertility level of an individual sire [3, 5].

Considering the biological and economical importance of knowing the potential fertility of the AI-semen prior to

insemination, major efforts have been made worldwide to design in vitro method/s that could explore aspects of

sperm structure and function and be, due to their relation to fertility, applied to a sub-population of processed bull

semen in order to determine their potential fertility if inseminated to a female bovine.

Traditional Evaluation of Frozen-Thawed Bull Semen To warrant the fertility of frozen semen in cattle AI, and thus promote the efficient dissemination of desirable

genetic material from proven sires onto a female population, the cryopreserved ejaculates from AI bulls are solely

evaluated for their levels of post-thaw sperm motility. Evaluation is mostly done subjectively, by visual

assessment on a microscope equipped with phase-contrast optics, thus relying entirely on the ability and

experience of the operator. The method is simple, easy and quick and remains the parameter of choice to

determine the degree of sperm damage inflicted by the cryopreservation procedure, specially under industrial

conditions. Although it is reported that there is a significant relationship between subjectively assessed motility

and field fertility [6]; such relationship is not strong (particularly when motility values are within ranges around 50%

or above) [7] and not always found in different cattle populations [8,9]. More and more bull stations are now

incorporating Computer-Assisted Semen Analyzers (CASA), aiming at a higher objectivity and the analyses of

certain patterns of sperm motility. Linearity, for instance, appears significantly correlated with field fertility [10,11].

ATP-determinations by luminometry can indirectly measure the number of viable spermatozoa, more accurately

than by visual assessment of sperm motility [12] but does not correlate to AI-fertility [4]. Sperm morphology

evaluation is a major component of the spermiogramme and indicates testicular and epididymal pathologies [9,13].

It can also provide some clues regarding the ability of spermatozoa to sustain freezing-thawing such as

membrane (acrosomes) and tail damage (single bent tails) [14] and thus let us refrain from using a certain

processed semen. New Approaches for Evaluation of Bull Frozen-Thawed Semen As mentioned above, bull spermatozoa have a well-defined structure specially differentiated for achieving

fertilization of the oocyte. The assessment of the integrity and functionality of different sperm parameters,

considered pre-requisites for fertilization due to their role in the interaction sperm-genital tract or sperm-oocyte [8]

has, therefore, been a priority. Evaluation of Sperm Viability The integrity of the plasma membrane has been one of the most studied parameters, for its major role in acting as

cell boundary and in cell-cell interactions, both in terms of morphological integrity as well as for its functional

intactness (Reviewed by 15]. Morphological evaluation, either using optical enhancement (as with differential

interference contrast optics; Nomarski), staining (supravital stains, such as fast green/eosin, eosin/aniline blue,

trypan blue/giemsa or naphtol yellow/erythrosin) for light microscopical examination or both scanning (SEM) and

transmission (TEM) electron microscopes has been of value. However, most of these techniques provide either

partial information or are time consuming, expensive, or both. Furthermore, even when some morphological

techniques provide insight into details of the plasma membrane injuries they are not correlated with the fertility of

the sire, unless major damage is present. The frequency of frozen thawed bull spermatozoa with intact plasma

membranes can be easily determined using simple and practical osmotic resistance tests (ORT) based up on

their behaviour when exposed to hypo-osmotic solutions [16,17]. Unfortunately, the outcome of the assay does

not always correlate with the fertility of the samples investigated. A major breakthrough to functionally assess

frozen-thawed bull spermatozoa has been the development of fluorescent probes for DNA, intracytoplasmic

enzymes, lectins or membrane potential [15]. Particularly the use of fluorophores (as single or combinations of

probes] have proven highly valuable to determine the integrity of the various subcellular sperm compartments

(mitochondrial function [Rhodamine 123], plasmalemmal integrity [fluoresceins, DNA-markers]). Fluorophores

have been used in connection with operator-screened fluorescent microscopy. Although inexpensive, only a few

hundred spermatozoa are assayed per sample, making this technique less accurate than flow cytometry (FACS

Analyzers), a technology that examines thousands of spermatozoa within minutes, but whose high cost still

represents a hindrance for its application in AI enterprises. As an alternative to assess sperm viability, fluorometry

can be used to read fluorescence in large sperm numbers per sample, being sufficiently accurate and rather

quickly and could, therefore, be applied for routine evaluation of semen quality [11]. The assessment of

membrane integrity in large sperm populations is reflected in a relation to fertility (albeit of low significance, 11], a

relation that was not present when fluorescent microscopy was used [18]. The integrity of the acrosome post-thaw

can also be determined morphologically, usually at the light microscopical level, in unstained samples or with

different empirical stains (Giemsa being among the most often used), with acrosome status being retrospectively

and significantly related to the fertility of frozen-thawed spermatozoa [19]. Fluorophores or lectins have also been

applied at this level with good correlations with fertility [20,21]. Acrosome integrity post-thaw in the bull can also

be assessed indirectly by measuring enzyme leakage (such as amidases, acrosin or lactic dehydrogenases [22]. Evaluation of Sperm Chromatin Status Concerning sperm chromatin status, evaluation of the degree of DNA denaturation using flow cytometry (the so-

called SCSA method, 23) appear to be a valuable complement for the routinely practiced microscopic evaluation

of sperm morphology of semen from bulls in regular production schedules, since some of the parameters tested

relate to bull fertility [11]. Relationship with fertility after AI As seen above, few single sperm viability parameters show a significant relation with the fertility of the assayed

frozen-thawed semen sample, specially if it lies within accepted ranges of normality [24]. Therefore, functional in

vitro tests e.g. able to disclose the ability of frozen-thawed spermatozoa to undergo complicated processes as

capacitation, binding to the zona pellucida (ZP), acrosome reaction, to fertilize oocytes (IVF), and to induce

embryo development in vitro have been designed and explored for their relation with the fertility achieved after AI. Assessment of Capacitation-Like Changes and the Induction of the Acrosome Reaction Sperm capacitation (and acrosome reaction) can be indirectly visualized by incubation of spermatozoa with the

antibiotic chlortetracycline (CTC), which fluoresces while monitoring Ca++ displacement in the sperm head

membrane. Analyses of frozen-thawed bull spermatozoa confirmed previous findings that cryopreservation

induces capacitation-like changes [2], which in AI-bulls with known fertility occurred in up to 30 - 40 % of their

processed spermatozoa [25]. Furthermore, the percentage of uncapacitated (unreacted) spermatozoa in an AI-

semen batch correlated positively with its fertility after AI [25]. The acrosome reaction (AR) could be induced in

vitro by exposure to glycosaminoglycans (GAGs) such as heparin [21,26]. The degree of AR after exposing frozen-thawed bull spermatozoa to heparin was significantly correlated with in vivo fertility [21]. Acrosome-

reactions can also be induced by treatment with calcium ionophores (as the Hoescht A23187), and significant

correlations have been found between the degree of induced AR and the NRRs of the bulls [27] even to a

predictive status [20]. Sperm Ability to Bind to the Zona Pellucida (Sperm-ZP Binding Assays, ZBA) Two types of sperm-ZP binding assays have been proven for bull spermatozoa, one using intact (not cleaved)

homologous oocytes [28,29] and the other using bisected hemizonae (hemizona binding assay, HZA) [30], where

each matching half is incubated with test vs control spermatozoa, respectively. Significant correlations with AI-

fertility have been found using both types of ZP-binding assays [30,31] (Fig. 1). The latter method (ZBA) appears,

however, much easier, less time-consuming and accurate than HZA, provided that a large number of oocytes are

included per test [29].

Figure 1. Correlation (r=0.50, P=0.02, n=22) between the mean numbers of bull spermatozoa bound to the ZP and the fertility (56-d NRR) of the bulls tested (modified, from 31). The inner lines mark a threshold of fertility and

a median of sperm numbers bound. Relationship Between IVF-Embryo Development and AI-Fertility The use of spermatozoa from a given bull affects IVF results, albeit a relationship between in vitro and in vivo

fertility was not always found [32-39]. Retrospective studies of frozen-thawed semen from bulls with a large range

of AI-fertility have demonstrated a significant positive correlation between cleavage rates in vitro and 56 d-NRRs

[38] (Fig. 2). Pursuing a later end point of the IVF (e.g. blastocyst production), a correlation with NRR decreased

owing to the higher dependence of the early embryo development on the conditions of culture than on the sperm

source.

Figure 2. Relationship between percentages of cleaved oocytes 48 h post-IVF and AI-fertility (56-d non-return rates, NRR), 2-4 freezing batches/bull, 15 bulls (modified, from 38).

Prognostic value of the in vitro tests As stated above, few single sperm parameters assessed in vitro relate to in vivo fertility. However, combining the

outcome of some sperm assays in a multiple regression analysis has shown that some of these provide a

discriminative, albeit retrospective, relationship with fertility [8,11,38,40] (Fig. 3). Despite this, correlation analyses

are retrospective and not prospective, that is they are not predictive per se. In vitro assays including ZP-binding

and IVF as above mentioned were used to calculate an expected fertility for frozen-thawed semen from young AI

bulls e.g. the different outcomes were combined to calculate corrected NRRs before the actual field fertility was

known [41]. When the actual 56d-NNRs for the young AI-bulls were later obtained, the calculated NNRs showed a

strong relationship with AI-fertility (Fig. 4), indicating it was possible to use a combination of laboratory tests to

determine semen quality and to give a prognosis on the potential fertility of an AI-bull. This appears to be an

effective tool for screening semen from young AI-bulls aiming to exclude those (see Fig. 4) with a potential lower

fertility from further progeny testing in an AI-program, thus allowing the restricted fertility and progeny testing

space available for young bulls to be used more efficiently, with obvious economic savings.

Figure 3. Relationship between NRRs predicted by the equation based on combination of four sperm parameters (post-thaw sperm motility, linear motility (CASA), frequency of spermatozoa with denatured DNA (SCSA) and

percentage of damaged spermatozoa (fluorometry) assessed in vitro and the observed field fertility (56-d NRR) of the batches tested (r=0.75, P<0.001). Line shows the trend in the data (from 11).

Figure 4. Predictive relationship (r=0.944, P=0.0001) between in vitro predicted NRRs (calculated from the testing in vitro of 3 frozen batches/bull combining 7 sperm parameters/batch) and the actual bull total AI-fertility (as 56d-NRRs). When the cut-off of sub-fertility was set at 62 % (lines) the prognosis revealed a single clear case, with

most other bulls at a similar level of fertility (around 65%) (modified, from 41). Acknowledgements The author's own studies have been supported by grants from the Swedish Council for Forestry and Agricultural

Research (SJFR), and the Swedish Farmer's Foundation for Agricultural Research (SLF), Sweden.

Sexing Bull Sperm

D. L. Garner and G. E. Seidel Jr

XY, Inc., Animal Reproduction and Biotechnology Laboratory, Colorado State University Foothills Research

Campus, Fort Collins, Colorado, USA.

College of Veterinary Medicine and Biological Sciences, Colorado State University, Fort Collins, Colorado, USA

Sexing Bull Sperm The sex of calves now can be predetermined with 85 - 95% accuracy [1,2]. This is accomplished by sorting sperm

with a flow cytometer/cell sorter. This technique, which was developed for live sperm by Dr. Larry A. Johnson at

the USDA Beltsville Agricultural Research Center, is patented [3-6] and exclusively licensed worldwide for non-

human mammals to XY, Inc., a private company. This company has invested heavily into advancing this

technology to the point that it has become a commercial reality [7,8]. Sperm Sexing Process Semen can be sexed because X-sperm, which produce heifers, contain 3.8% more DNA than Y-sperm, which

produce bull calves [5]. Freshly collected sperm are stained with a specific bisbenzimidazole DNA-binding dye,

Hoechst 33342, for 1 h [9]. The stained sperm fluoresce bright blue when exposed to a laser beam of short

wavelength light. The stained X-sperm emit a brighter fluorescence than Y-sperm because of the greater DNA

content. This difference in emitted fluorescence can be measured by a photomultiplier tube (PMT) and integrated

using a powerful computer so that DNA content for most, but not all sperm can be accurately determined as they

pass the PMT in a stream of fluid. As the fluid stream containing the sperm exits the nozzle of the sorter, it is

vibrated at a high frequency causing individual droplets to form at a rate of about 90,000/sec [9]. Although not all

droplets contain sperm, those that do are given a positive or negative charge, depending on the DNA content

information that was provided by detector. Sperm must be oriented properly to measure DNA content accurately

[5]. No charge is applied to droplets containing more than one sperm, dead sperm as determined by uptake of a

vital dye, or those sperm where DNA content could not be measured accurately, thereby simply allowing these

cells to be disposed of as waste. Charged droplets containing X-sperm, which had been negatively- or positively

charged according to sperm DNA content, are deflected by an oppositely charged plate thereby directing the

sperm into a collection vessel. Droplets containing Y-sperm are simultaneously directed to a different collection

vessel by applying an opposite charge to those drops so that the sperm are deflected toward an opposing-

charged plate. Thus, the streams of droplets containing the X-sperm, the Y-sperm or no sperm or too many sperm

are collected into three separate vessels. This process allows sexing and collection of about 40% of the sperm

going through the sorter at a speed of approximately 100 km/h. Thus, at an event rate of 20,000 total sperm/sec,

nearly 4,000 live sperm/sec of each sex can be sorted simultaneously [9]. The current system can produce

approximately 10 to 13 x 106 live sperm/h of each sex with 85 - 95% accuracy. Low-Dose Insemination The sorting process dilutes sperm, so they need to be re-concentrated by centrifugation prior to packaging in 0.25

ml French straws at doses of 1 to 6 x 106 sperm/straw. Conventional artificial insemination (AI) procedures utilize

around 20 x 106 sperm/straw, so the insemination dose of sex-sorted sperm is about 1/20 to normal, frozen-

semen AI dose. Even at the achieved superior sort rates, it is necessary to use 1/3 fewer sperm per insemination

than used conventionally to have a commercial product [10]. In recent work in Holland, normal non-return rates

were achieved with less than 2 x 106 sperm when semen from highly fertile bulls was used [11]. Utilization of only

the top 20% of fertile bulls would certainly expedite implementation of sexed sperm. Low-dose insemination

allows many more heifers to be bred with sex-sorted sperm than with current AI practices. Under the current

system for sexing, bovine sperm are cryopreserved so that they can be efficiently used for AI of estrus-

synchronized heifers. The inherently higher fertility of heifers makes them more suitable than cows for the current

sexing technology. Preliminary results using low-doses of sex-sorted sperm for breeding lactating cows have

been less than satisfactory [1].

Estrus Synchronization Optimal results can be obtained only if the heifers to be inseminated are properly managed [1]. Several

successful regimens have been used to synchronize estrus for sexed sperm including 1) 500 mg of melengestrol

acetate (MGA) fed daily in grain for 14 d followed by an IM injection of 25 mg prostaglandin F2α 17, 18 or 19 d

after the last day of feeding MGA; 2) a single injection IM of 25 mg prostaglandin F2α; 3) 20 or 25 mg

prostaglandin F2α injected IM at 12-d intervals; and 4) 50 or 100 µg of GnRH injected IM followed by 25 mg of

prostaglandin F2α 7d later. Insemination Heifers were inseminated after 16:00 h so that the insemination occurred approximately ½ or 1 d following the

onset of estrus [1], with no significant difference in pregnancy rates with sexed sperm between these times (45

and 49%, respectively). Straws containing the sex-sorted sperm were thawed for 20 to 30 sec in a 34 to 37°C

water bath before being immediately inseminated at either of two sites, into the lumen of the uterine body, as is

done for conventional AI, or deep in the uterine horn [1]. Between May 1998 and July 1999, more than 1,000

heifers were bred by 7 inseminators using sex-sorted, cryopreserved sperm from 22 bulls of unknown fertility

representing various dairy and beef breeds [1]. Pregnancy Rates Small, non-significant differences in pregnancy rates were noted between 1.0 to 1.5 x 106 vs. 3.0 x 106 sexed,

cryopreserved sperm for the 1,000 inseminations described above [1] (Table 1). Furthermore in some of the later

trials of this group, the pregnancy rates for sexed, cryopreserved sperm sometimes were 90% of controls that had

7 to 20 times more sperm/insemination dose [1]. In some early trials, pregnancy rates for controls were much

higher than for sexed sperm. Only in one trial did inseminations into the uterine horn result in higher pregnancy

rates than when the sperm were placed in the uterine body as is done for conventional AI [1]. No differences were

noted among inseminators. Some bull differences were suggested, but numbers of inseminations per bull were

inadequate to draw any firm conclusion.

Table 1. Summary of trials with sexed, frozen sperm and frozen controls [1]

Sperm no./site No. heifers No. pregnant

1.0-1.5 x 106/body 176 98 (56%)

3.0 x 106/body 171 88 (51%)

20 x 106/body, control 183 124 (68%)

1.0-1.5 x 106/body 163 70 (43%)

1.0-1.5 x 106/horn 158 85 (54%)

20 x 106/body, control 128 79 (62%)

Calving Results Not all animals were followed to calving due to the need for collaborators to market the animals as “springer”

heifers. Several hundred heifers have been followed to term and many more are currently gestating. In those

animals that were followed to term, there did not appear to be any excess embryonic deaths between 1 and 2

months of gestation, and very few abortions occurred between 2 months gestation and term [1]. Rigorous

epidemiological studies, however, need to be done to confirm that calves from sexed sperm are completely

normal. Recently, three heifers that were produced from sexed sperm calved as a result of being themselves

inseminated with sexed sperm. This second generation of calves from sexed, cryopreserved sperm demonstrates

the feasibility of using an all heifer production system, whereby heifers are bred with sexed sperm to produce their

own replacements [12].

In Vitro Fertilization (IVF) Although the first sexed-selected calves were born from embryos derived from IVF with sex-sorted sperm [13],

most of the recent work has been done using AI [1,14]. Large-scale trials are now underway to examine the

normalcy of calves born from IVF-produced embryos. Summary Commercialization of sexed sperm for AI in heifers is imminent.

Scrotal/Testicular Thermoregulation in Bulls

J. P Kastelic, R. B. Cook and G. H. Coulter

Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge Alberta, Canada.

Summary Testicular temperature in bulls must be 2 to 6 degrees Celsius cooler than core body temperature for the

production of fertile spermatozoa. Mechanisms that cool the testes include the testicular vascular cone (functions

as a counter-current heat exchanger), heat loss from the scrotal surface, relaxation of scrotal muscles, scrotal

sweating, whole-body responses, and complementary temperature gradients (in the scrotum and testes). The

testes operate on the brink of hypoxia. When testicular temperature increases, metabolism increases at a greater

rate than blood flow and hence the testes become hypoxic. Therefore, the testes are very susceptible to

temperature increases due to endogenous or exogenous factors (e.g. fever, high ambient temperature). As

testicular temperature increases, the proportion of defective spermatozoa increases; recovery is dependent upon

the nature and duration of the thermal insult. Introduction Bull fertility is of paramount importance in cattle production; one bull may be exposed to 20 females under natural

service conditions or hundreds of thousands via artificial insemination. Although few bulls are sterile (unable to

reproduce), there is a large range in bull fertility, especially in unselected populations [1]. It is well known that

testicular temperature in bulls must be 2 to 6 degrees Celsius below core body temperature for the production of

fertile spermatozoa and that increases in testicular temperature decrease semen quality [2]. Increased testicular

temperature is the underlying cause of infertility in many bulls. Anatomy and Physiology Several features contribute to the regulation of testicular temperature. The pampiniform plexus is a complex

venous network surrounding the highly coiled testicular artery; the entire structure (venous network and artery) is

properly called the "testicular vascular cone" [3]. In the testicular vascular cone, arterial blood is cooled as heat is

transferred from the artery to the vein in a classical counter-current heat-transfer system. Furthermore, this is an

important site of surface heat loss as the skin overlying the cone is usually the warmest area on the scrotum [4].

Characteristics of the testicular vascular cone and scrotal surface temperature in bulls from 0.5 to 3 years of age

have been reported [5].

Scrotal skin is usually thin and relatively hairless. There is an extensive subcutaneous blood and lymphatic

system, with blood vessels located superficially, facilitating heat transfer [6]. Smooth muscle in cutaneous

arterioles of the scrotum are innervated by sympathetic neurons [7]; stimulation of these neurons causes

vasoconstriction [7]. An increase in scrotal temperature causes dilation of these arterioles by direct action of heat

and reflex removal of sympathetic vasoconstrictor tone [8].

The scrotal neck is the warmest part of the scrotum [4]; a long, distinct scrotal neck (pendulous scrotum) provides

a large area for heat loss and moves the testes away from the abdomen. The tunica dartos, a thin sheet of

smooth muscle under the scrotal skin, is controlled by sympathetic nerves and contracts and relaxes in cold and

warm environments, respectively [8]. The cremaster muscle also contracts to move the testes closer to the body

in cold conditions; however, unlike the tunica dartos, it is a striated muscle and cannot sustain contraction for

prolonged intervals [8].

Sweating and whole-body responses contribute to testicular cooling and have been best characterized in sheep.

In Merino rams, scrotal sweat glands are larger and produce more sweat than those elsewhere on the body [9].

Similarly, sweat gland density is higher in scrotal skin than any other body region in bulls [10]. Apocrine sweat

glands in the scrotum of rams discharge simultaneously; expulsion begins when scrotal surface temperature is

about 35.5 degrees Celsius and occurs at a frequency of up to 10 discharges per hour [11]. Whole-body response

in rams includes an increase in respiration rate when scrotal surface temperature rises above 35 - 36 degrees

Celsius [8]. Furthermore, when scrotal surface temperature in rams reaches 38 - 40 degrees Celsius, respiration

becomes very rapid (e.g. 200 breaths per minute], there is peripheral vasodilation and temperatures within the

rectum and carotid artery can decline as much as 2 degrees Celsius in 1 hour [12]. Surface and Internal Temperatures In 16 crossbred beef bulls [13], temperatures were measured at three locations in each testis: top, middle and

bottom. Average temperatures (degrees Celsius) at these locations were: 30.4, 29.8 and 28.8 (scrotal surface

temperature); 33.3, 33.0 and 32.9 (scrotal subcutaneous temperature); and 34.3, 34.3 and 34.5 (intratesticular

temperature). Top-to-bottom temperature gradients were 1.6, 0.4 and -0.2 degrees Celsius for scrotal surface,

scrotal subcutaneous and intratesticular temperatures, respectively. Therefore, the temperature gradient was

most pronounced on the scrotal surface, small in the scrotal subcutaneous tissues, and absent within the

testicular parenchyma. It was subsequently shown that the scrotal surface and testes have opposing,

complementary temperature gradients, resulting in a relatively uniform intratesticular temperature [14].

Furthermore, although intratesticular temperature was significantly higher when a testis was within the scrotum

compared to when it was exposed, scrotal surface temperature was similar whether or not there was an

underlying testis [14]. Therefore, the scrotum has a significant influence on testicular temperature but the testes

have a small influence on scrotal temperature.

Scrotal and testicular temperature gradients may be due to vasculature. The scrotum is vascularized from top to

bottom. However, the testicular artery (after exiting the ventral aspect of the testicular vascular cone) courses the

length of the testis (under the corpus epididymis), reaches the bottom of the testis, and then diverges into multiple

branches that spread dorsally and laterally across the surface of the testis before entering the testicular

parenchyma [15]. Therefore, the testis is vascularized from the bottom to the top. In a recent study [16] it was

shown that blood within the testicular artery has a similar temperature at the top of the testis (below the testicular

vascular cone) compared with the bottom of the testis, but was significantly cooler at the point of entry into the

testicular parenchyma (intra-arterial temperatures 34.3, 33.4 and 31.7 degrees Celsius, respectively).

Consequently, these opposing temperature gradients collectively result in a nearly uniform intratesticular

temperature.

In bulls, temperatures of the caput, corpus and cauda epididymis averaged 35.6, 34.6 and 33.1 degrees Celsius,

respectively, and the gradient between the caput and the cauda averaged 2.5 degrees Celsius [13]. The

temperature of the caput was greater than that of the testicular parenchyma at the top of the testis, probably

because the caput is close to the testicular vascular cone. However, the cauda, an important site for sperm

storage and maturation, was slightly cooler than the testicular parenchyma. Sources of Testicular Heat Testicular blood flow and oxygen uptake were recently measured in eight Angus bulls to determine the

importance of blood flow versus metabolism as sources of testicular heat [17]. Blood flow in the testicular artery

averaged 12.4 mL per minute. Arterial blood was warmer (39.2 versus 36.9 degrees Celsius, P<0.001) and had a

higher percentage of hemoglobin saturated with oxygen than blood in the testicular vein (95.3 versus 42.0%

P<0.001). Based on blood flow and hemoglobin saturation, the oxygen used by one testis (1.2 mL per minute)

was calculated to produce 5.8 calories of heat per minute, compared to 28.3 calories per minute attributed to

blood flow (approximately a five-fold difference).

The testis usually operates on the brink of hypoxia [8]. Increased temperature increases metabolism, with a

concurrent need for increased oxygen to sustain aerobic metabolism. However, studies in rams [8] have shown

that blood flow changes little in response to increases in testicular temperature and consequently the testes

become hypoxic. Increasing blood oxygen saturation is not practical since the blood is nearly completely

saturated under normal conditions. Although increasing blood flow would increase the delivery of oxygen, it would

also bring considerable additional heat into the testes. Therefore, increasing heat loss from the scrotum would

appear to be the most appropriate response. Evaluation of Scrotal Surface Temperature with Infrared Thermography Infrared thermograms of the scrotum of bulls with apparently normal scrotal thermoregulation had left-to-right

symmetry and were 4 to 6 degrees Celsius warmer at the top of the scrotum than at the bottom [4,18]. More

random temperature patterns, often lacking left-to-right symmetry and having localized areas of increased

temperature, were interpreted as abnormal thermoregulation of the testes or epididymides. Although bulls with

abnormal thermograms usually had poor quality semen [4,18], not every bull with poor quality semen had an

abnormal thermogram. In bulls with unilateral orchitis, the scrotal surface temperature was greater over the

affected testis compared to the other testis [18]. Scrotal surface temperature in rams [19] was highly correlated

with both scrotal subcutaneous temperature and with the temperature of a surrogate testis (water-filled balloon).

However, it is now recognized that caution must be exercised when making inferences about intratesticular

temperature based on measurement of scrotal surface temperature [13].

Infrared thermography has been used as an adjunct to the standard breeding soundness examination. Thirty

yearling beef bulls that were satisfactory on a standard breeding soundness examination, were individually

exposed to approximately 18 heifers for a 45-day breeding period [20]. For bulls with a scrotal surface

temperature pattern that was classified as normal or questionable, pregnancy rates 80 days after the end of the

breeding season were similar (83 versus 85%), but were higher (P<.01) than pregnancy rates for bulls with an

abnormal scrotal surface temperature pattern (68%). Effects of Increased Testicular Temperature Increased Ambient Temperature The effect of increased ambient temperature on semen quality has been determined in many studies. In one

study, two Guernsey bulls were exposed to 37 degrees Celsius and 81% relative humidity for 12 hours per day for

17 consecutive days [21]. Approximately 30 to 40% of the spermatozoa were morphologically abnormal (mostly

coiled tails and detached heads) and the total number of spermatozoa, sperm concentration, and motility

decreased profoundly. In another study [22], ambient temperatures of 40 degrees Celsius at a relative humidity of 35 to 45% for as little as 12 hours reduced semen quality. Bos taurus bulls are more susceptible to high ambient

temperatures than Bos indicus bulls [22]. Following exposure to high ambient temperatures, decreases in semen

quality were less severe, occurred later and recovered more rapidly in crossbred (Bos indicus x Bos taurus) bulls

than in purebred Bos taurus bulls [23].

Scrotal Insulation Insulation of the scrotum (with cloth, wool or other materials) has been frequently used as a model of increased testicular temperature. In one study [24], the scrotum of Bos indicus x Bos taurus bulls was insulated for 48 hours.

The nature and time (Day 0 = initiation of insulation) of morphologically abnormal spermatozoa that resulted were:

decapitated, Days 6 - 14; abnormal acrosomes, Days 12 - 23; abnormal tails, Days 12 - 23; and protoplasmic

droplets, Days 17 - 23. Therefore, scrotal heating affected spermatozoa in the caput epididymis as well as

spermatids. Although daily sperm production was not affected, epididymal sperm reserves were reduced by

nearly 50% (9.2 billion versus 17.4 billion), particularly in the caput (3.8 vs 6.6 billion) and cauda (3.7 versus 9.5

billion), perhaps due to selective resorption of abnormal spermatozoa in the rete testis and excurrent ducts. In

another study [25,26], the scrota of six Holstein bulls was insulated for 48 hours (Day 0 = initiation of insulation).

The number of spermatozoa collected was not significantly affected but the proportion of progressively motile

spermatozoa decreased from 69% (prior to insulation) to 42% on Day 15. The proportion of normal spermatozoa

was not significantly different from Day -6 to Day 9 (80%), decreased abruptly on Day 12 (53%) and reached a

nadir on Day 18 (14%). Although there was considerable variation among bulls in the type and proportion of

abnormal spermatozoa, specific abnormalities appeared in a consistent chronological sequence: tailless, Days 12

to 15; diadem, Day 18; pyriform and nuclear vacuoles, Day 21; knobbed acrosome, Day 27; and Dag defect, Day

30. When spermatozoa were collected 3-9 d after insulation and examined immediately, their motility and

morphology were similar to pre-insulation values [25]. Compared to semen collected prior to insulation, following

freezing, thawing and incubation at 37 degrees Celsius for 3 hours [25], there were significant reductions in the

proportion of progressively motile spermatozoa (46 versus 31%, respectively) and the proportion of spermatozoa

with intact acrosomes (73 versus 63%). Freezing plus post-thaw incubation manifested changes that had

occurred in spermatozoa that were in the epididymis at the time of scrotal insulation.

In a recent study [27], scrotal insulation (4 days) and dexamethasone treatment (20 mg per day for 7 days) were

used as models of testicular heating and stress, respectively. Some bulls seemed predisposed to produce

spermatozoa with a particular abnormality. Pyriform heads, nuclear vacuoles, microcephalic sperm, and abnormal

DNA condensation were more common in insulated than dexamethasone-treated bulls. Conversely,

dexamethasone treatment resulted in an earlier and more severe effect on epididymal spermatozoa, an earlier

and greater increase in distal midpiece reflexes, and an earlier increase in proximal and distal droplets. In general,

the types of defective spermatozoa and the time of their detection were similar for the two treatments. Insulation of the Scrotal Neck The scrotal neck of five bulls was insulated for 7 days (Days 1 to 8) as a model of bulls with a excessive body

condition (that usually have considerable fat in the neck of the scrotum). Spermatozoa within the epididymis or at

the acrosome phase during insulation appeared to be most affected [28]. Insulated bulls had twice as many

spermatozoa with midpiece defects and four times as many with droplets on Day 5, fewer normal spermatozoa

and three times as many with midpiece defects and droplets on Day 8, fewer normal spermatozoa on Days 15

and 18, and more spermatozoa with head defects on Days 18 and 21. Semen quality in insulated bulls had nearly

returned to pre-insulation values by Day 35. In a second experiment [28], scrotal subcutaneous temperature

increased 2.0, 1.5 and 0.5 degrees Celsius at the top, middle and bottom of the testis, respectively, and

intratesticular temperature was 0.9°C higher at the corresponding three locations 48 hours after scrotal neck

insulation compared to pre-insulation. Clearly the scrotal neck is an important site of heat loss. Increased Epididymal Temperature In the majority of animals, the cauda epididymis is somewhat cooler than the testes [29], facilitating its sperm

storage function. Increasing cauda temperature disrupts the normal absorbtive and secretory functions, changes

the composition (ions and proteins) of the cauda fluid, and increases (approximately three-fold) the rate of sperm

passage through the cauda [29]. Consequently, the number of sperm in the first ejaculate declines, with an even

more dramatic decline in sperm number in successive ejaculates. In addition, the increased temperature seems to

prematurely hasten sperm maturation [29]. Effects of Increased Temperature on Testicular Cells Although heating seems to affect Sertoli and Leydig cell function, germ cells are the most sensitive to heat [30].

All stages of spermatogenesis are susceptible, with the extent of damage related to the extent and duration of the

increased temperature [30]. Spermatocytes in meiotic prophase are killed by heat, whereas spermatozoa that are

more mature usually have metabolic and structural abnormalities [31]. Heating the testis usually decreases the

proportion of progressively motile and live spermatozoa, and increases the incidence of morphologically abnormal

spermatozoa, especially those with defective heads [32]. Although there is considerable variation among bulls in

the nature and proportion of defective spermatozoa, the order of appearance of specific defects is relatively

consistent [26,27]. Unless spermatogonia are affected, the interval from cessation of heating to restoration of

normal spermatozoa in the ejaculate corresponds to the interval from the beginning of differentiation to ejaculation

[30]. Even though sperm morphology has returned to normal, their utilization may result in decreased fertilization

rates and an increased incidence of embryonic death [33]. Summary of Increased Testicular Temperature When scrotal/testicular temperature is increased (regardless of the cause), sperm morphology is generally

unaffected initially (for an interval corresponding to epididymal transit time) but subsequently declines [32]. In

some studies [24,28], spermatozoa that would have been in the epididymis at the time of scrotal heating were

morphologically abnormal when collected soon after heating. In another study [25], changes in these

spermatozoa were manifest only after they were frozen, thawed and incubated. Sperm morphology usually

returns to pre-treatment values within approximately 6 weeks of the thermal insult. However, a prolonged and (or)

severe increase in testicular temperature will increase the interval for recovery. It appears that the decrease in

semen quality associated with increased testicular temperature is ultimately related to the severity and the

duration of the increased testicular temperature.

Management and Economics of Natural Service Sires on Dairy Herds C. A. Risco

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Introduction Reproductive efficiency in a dairy herd improves when the percentage of pregnancies resulting from artificial insemination (AI) increases. As described by Bartlett's equation of reproduction, four factors determine the percent of resulting pregnancies from AI [1]. These factors are; cows detected in heat and inseminated, fertility level of the herd, semen fertility level and inseminator efficiency. The percentage of pregnancies resulting from AI is the product of these four factors and not their average. When these factors are multiplied, their product or percent pregnant is less than the lowest factor. The effect of a low factor has a cumulative effect on the percentage of pregnancies and is never averaged out. Examining this equation provides producers with a working concept of dairy cattle reproduction. Of the factors included in the equation of reproduction, the inability to detect estrus efficiently and accurately is the major impediment in attaining an optimal percentage of pregnant cows from AI. Estrus detection efficiency is expressed as the percentage of estruses observed over a given time period [2]. Accuracy of heat detection is the percentage of estruses observed that are true estruses [2]. Collectively, errors in efficiency and accuracy of heat detection result in high semen cost and an increase in the interval from calving to conception, reducing cow production and net returns. To eliminate inefficient heat detection practices the use of natural service (NS) bulls in dairy farms appears to be increasing. The perception is that pregnancy rates improve when NS is used because more cows are detected in true estrus and serviced; that is the intensity and accuracy improves in the herd. A 1984 survey of dairy herds in Florida showed that 50 per cent used AI, 38 per cent used a combination of AI and NS, and the remainder used mostly NS [3]. A Pennsylvania study involving 329 dairy farms, evaluated the method used in breeding heifers. Here, 11.2 per cent bred their heifers once with AI then used a bull, 8.5 per cent bred their heifers twice with AI then used bulls and 20.7 per cent bred their heifers with bulls only [4]. Estimates from large dairy herds in Florida, and Texas indicate that the use of NS is still widespread particularly in dairy herds employing a rotational grazing management. A 1995 survey from the National Association of Animal Breeders reported that less than twenty per cent of dairies use artificial insemination exclusively [5]. Dairy herds that predominantly use NS bulls usually do not raise replacement heifers; the genetic balance of the herd can be maintained by purchasing replacement heifers from breeders who are using AI with semen from proven bulls. The use of NS can reduce the negative effect that people can have on cattle reproduction by eliminating errors in estrus detection. However, when the equation of pregnancy rate is considered using NS, it becomes apparent that the fertility of the bull, and his ability to service cows that are in estrus greatly determines the pregnancy outcome. Therefore, in order to adequately exploit the use of NS in dairy herds, proper selection and management of these bulls should be included in the overall herd health program of the dairy. In addition, to prevent the disastrous economic consequences of sub-fertile bulls, periodic evaluation of their reproductive performance must be performed.

Economics of Heat Detection (AI vs NS) The economics of NS bulls versus AI are usually determined by calculating the cost of semen, equipment, personnel and cost of bull maintenance. The major argument against the use of NS is the predicted difference in milk yield of AI daughters compared to NS daughters [3]. However, a major economic consideration for using NS bulls is the potential for the improvement in the herds reproductive efficiency by maximizing heat detection and conception rates. Use of NS bulls may result in higher conception rates when compared to AI because errors in heat detection are removed. In general, as heat detection rates improve, net revenues increase as a result of higher milk yields per cow. To illustrate this concept, the projected effect of three different heat detection rates on milk production and economic return is shown in Table 1 [3]. The model uses a seasonally adjusted conception rate of 30 per cent [6]. This model shows that an increase in heat detection rate from 47 per cent to 57 per cent produces an increase in milk yield of 370 lb of milk per cow per year and $39.97 per cow per year when modeled over a 10 year period. In addition, the replacement rate of a herd is also reduced as heat detection rates improve (Table 2) [3].

Table 1. Estimated long-term (10 yr) effects of heat detections rates (47, 57, & 67%) on milk production & net revenues/cow/yr at a seasonally adjusted conception rate of 30% (adapted from [3], p 209).

Heat Detection Rate

Annual Milk Production/cow (10 yr avg)

Excepted Change per +10% HDR

Net Revenues/Cow/Year

(10 yr avg.)

47 14,914 lb --- 700.82

57 15,284 lb +370 lb 740.75

67 15,476 lb +192 lb 763.72

Table 2. Culling/replacement rate and average months in the herd/cow when conception rate averages 30% and heat detection rate is varied (47, 57, & 67%) (adapted from [3], p 210).

Heat Detection Rate

47% 57% 67%

Replacement Rate 0.41 0.35 0.33

Avg. Months in Herd/Cow 25.06 28.08 29.54

By evaluating the prevailing heat detection and conception rates in a herd, the effective pregnancy rate (heat detection rate x conception rate) of that herd can be determined. The effective pregnancy rate can then be used to determine if the use of NS bulls would improve reproductive efficiency in the herd. Pregnancy rate exert their greatest effect on herd production, profit and replacement rate when it is between 25 and 30 per cent (Table 3) [3]. Each percentage point increase in effective pregnancy rate between 15 to 25 per cent, gives an increase of 1301 lb/cow/yr and $140 net/cow/yr. Between 25 and 35 percent, each percentage point of effective pregnancy rate is worth 355 lbs/cow/yr and $37 net/cow/yr. Conversely, if a dairy herd has an effective pregnancy rate greater than 25 per cent, the use of NS bulls would not result in a significant improvement in net income per cow. Therefore, the expected change in yield per cow based on increased heat detection, through the use of NS bulls or improved heat detection management, can be utilized to calculated the net value of high predicted difference AI semen as compared with the value of higher heat detection rate [3].

Table 3. Effective pregnancy rate (conception rate x heat detection rate) influence on milk/cow/yr, net income/cow/yr, and replacement rate (adapted from [3], p 212).

Effective Pregnancy Rate Milk/Cow/Yr Net $/Cow/Yr Replacement Rate

0.15 14,826 $688 0.38

0.25 16,127 $828 0.27

0.35 16,482 $865 0.26

0.45 16,726 $896 0.25

The male effect, or biostimulation, on female reproduction is well known [7]. Rams introduced early in the breeding season induce, and appear to synchronize, estrus in ewes. In sheep this biostimulation effect occurs in ewes that are in transitional cyclicity from the non-breeding to the breeding season and the males are introduced as a novel stimulus. Natural service bulls are often used in dairy herds in cows which already have cycled and have received multiple AI. These are the so called repeat or problem breeders and in this scenario biostimulation would probably not play a role. However, in those herds that use natural service only, bulls are introduced to cows in early lactation when the cows are in a transition cyclicity state. In this situation it is possible that some form of biostimulation may occur. An advantage seen for NS over AI suggested a possible male effect [8]. It should be kept in mind that comparison of NS versus AI are difficult to make. Researchers in this field conclude that biostimulation effects in cattle due to bull presence remain unclear. In addition there is anecdotal evidence from private practitioners that NS bulls reduce ovarian cystic degeneration in dairy cattle.

Selection of Bulls Regardless of the genotype used for natural service in a dairy herd bulls must be carefully selected. The selected

bulls should have the capability to detect females in estrus and be able to impregnate them. The ability of the bull to perform this task is dependent of his semen quality, libido, mating ability and social ranking among other bulls and females [7]. Therefore, in common with the recommendations made for beef bulls, selection of bulls for natural service in dairy herds should be subjected to a breeding soundness examination, as recommended by the Society of Theriogenology [9]. Only those bulls that successfully pass their BSE should be used. This examination should be repeated on an annual basis. It is recommended to use younger bulls that are less than 2 to 2.5 years old [3]. Young bulls should have achieved full puberty and sexual maturity which occurs around 14 months of age and should not be under-sized in relation to mature cows. Economic losses that occur from use of NS bulls due to lower milk production in daughters from these bulls are high. The sire-of daughter pathway was the weakest area of genetic improvement in the United States because of extensive use of NS bulls with low genetic merit [10]. Lost revenue represents the value of 695 lb of milk in each lactation over each generation. To help reduce these losses from genetically inferior NS bulls, producers should consider using bulls for natural service that are good enough for AI sampling. The genetic merit of young bulls used in AI sampling is as good as that for the average active AI bull. The typical bull in AI sampling would be at percentile 50, similar to the typical active AI bull [10].

Measuring Efficiency of NS Bull Breeding Programs Adequate records and their proper analysis and interpretation are fundamental to effective reproductive management. Dairy herd improvement association (DHIA) records are widely used by dairymen throughout the U.S., and are frequently analyzed by veterinarians in North America [11]. In dairy herds that use NS bulls in their reproductive program, DHIA records can be used to evaluate the overall herd reproductive performance, which includes breeding for both AI and NS bulls. However, these records are not designed to access the efficiency or performance of NS bulls used in the herd. Therefore, accurate evaluation of the dairy's NS reproductive program is difficult to make. A case in point is the practice by many Dairy Records Processing Centers to enter just a single service in the record for successful bull breedings regardless of the number of services. This practice gives an inaccurate evaluation of NS bulls reproductive performance. It is important to accurately monitor NS bull performance in order to make correct and prompt decisions to replace sub-fertile bulls. The reproduction committee of the American Association of Bovine Practitioners in 1991 recommended reproductive indices for herds using NS bulls [12]. These indices have been summarized by Chenoweth and Larsen [3] and Upham [13].

1. Percentage Cows Pregnant by the Bull Calculated as:

BP/TP x 100 Estimates the percentage of pregnancies due to NS bulls (BP) relative to all pregnancies including AI in the herd (TP). This measurement requires that the veterinarian estimate the date of conception in cows that have been bred by a bull so that pregnancies from NS can be distinguished from AI. A high value indicates a low estrous detection and AI technician efficiency for the AI component of the herd [3]. 2. Average Days Open with the Bull Calculated as: sum of days between Turned with Bull date and estimated date of conception for cows confirmed in bull breeding

number of cows confirmed to bull breeding

A performance value recommended is between 40 to 50 days. Elevated values could indicate low cow fertility or low bull fertility [13].

3. Cow to Bull Ratio Calculated as:

Cows Turned with Bull and not confirmed pregnant

number of bulls with access to cows

This calculation is used to determined if the low bull fertility is caused by a large cow to bull ratio. The cow to bull ratio should vary between 20 to 30 [3,13].

4. Bull Services per Pregnancies Calculated as:

average of (conception date -[turned with bull date + 10]) for all cows confirmed pregnant to bull during period

21

This calculation excludes open exposed cows until they are diagnosed pregnant. The reciprocal of this figure estimates the conception rate for bull services and can be used for comparison with AI conception rates [13]

The above calculations require that diligent records be kept by the producer and analysis of these records are conducted on a timely basis. In those herds that use only NS and do not record when bulls are introduced to cows, the author has had success in monitoring bull fertility by monitoring per cent of cows pregnant at the end of a herd check and cows open >150 days in milk. Except for the summer months these figures should remain constant on a monthly basis. Accurate information on NS breeding efficiency is difficult to obtain because in many situation NS bulls are used with cows that have failed to conceive after various AI attempts [14].

Management of Bulls In many situations, NS bulls fail to participate in the health programs designed for the cows. Bulls used for NS should receive the same vaccinations for cows with the exception of Brucellosis. Venereal diseases such as vibriosis and trichomonosis are an important consideration when using NS bulls. For vibriosis vaccination of females affords the best protection with best timing at several weeks prior to breeding. It is recommended that cows are vaccinated at least 3 weeks prior to bull exposure. Some success also has been attained with the vaccination of bulls [3]. Vaccination for trichomonosis is also available for females only. The clinical picture for both of these venereal diseases is similar. The herd picture is one of repeat breedings which contribute to an increase to the interval from calving to conception in the herd. In addition, abortions may occur in the second trimester. In trichomonosis, pyometra may occur in some cows. Diagnosis is best done with appropriate sampling from both bulls and cows. In cows affected with pyometra as a result of trichomonosis, culturing is often unrewarding. In the majority of dairy farms bulls are purchased and have an unknown history of vaccination. It may be worthwhile for the veterinarian to design a protocol for the management and care of bulls used for NS that includes a physical examination, BSE, vaccination and deworming program. Bulls being used for natural service should not be allowed to become overconditioned or to develop feet and leg problems. These undesirable traits negatively affect the breeding soundness of bulls. Rations which are balanced for middle to high producing dairy cows contain higher energy, protein and calcium levels than those required by the bull [15]. The excess in energy intake can predispose the bull to overconditioning and laminitis. Feeding bulls a high level of dietary calcium has been associated with lameness in conjunction with bone lesions in the spine and hip regions [3]. The fact is that when one examines the dietary requirements for mature bulls regardless of genotype, they are similar to requirements of a dry dairy cow . To avoid problems related to a lactating cow ration, evaluation of body condition and lameness should be conducted frequently in NS bulls. During the past years there has been a concern over the effect of gossypol from diets containing cottonseed products on bull fertility. In many dairy regions of the United states as much as 8 pounds (15 per cent DM basis) of whole cottonseed is fed in total mixed rations balanced for high producing dairy cattle. A mature Holstein bull with an dry matter intake of 13 kg [15] could consume as much as 13 g of free gossypol per day. Whether or not gossypol intake at this level has a detrimental effect on bull fertility is not definitively known. An increase in sperm midpiece abnormalities and erythrocyte osmotic fragility in Brahman bulls fed 2.75 kg of cottonseed meal (8.2 g of free gossypol per day) has been reported [16]. In contrast, Hereford bulls ingesting 7.6 to 19.8 g of free gossypol daily from whole cottonseed showed no significant sperm cell abnormalities [17]. In the above study [16], it was suggested that the mineral content of the drinking water contained sufficient minerals to bind with the free gossypol. The type of cottonseed product (meal vs whole seed), and gossypol enantiomer (+ or -) may determine the extent of the toxicological effect that will occur and may explain the variable results obtained in research trials [18]. It has been suggested that detoxification of gossypol in the rumen is more efficient with whole seed diets than with cottonseed meal diets [18]. Brahman bulls fed 1.8 g/day of free gossypol from cottonseed meal had similar damage to seminiferous epithelium when compared to bulls fed 16 g/day of free gossypol from whole cottonseed [19]. The spermicidal effect of gossypol may also depend on the predominant + or - gossypol enantiomer present in the cottonseed product. Due to its stereospecific binding properties, the (-) gossypol enantiomer is less bound to plasma proteins and appears better able to cross the blood-testis barrier in vivo and inhibit the biological activity of some proteins [20]. The type of cottonseed used and gossypol enantiomer present, has contributed to the variable results obtained in gossypol related studies. Recommendations in terms of gossypol intake in the total diet for bulls used for breeding is 200 mg/kg for diets composed of cottonseed meal and 900 mg/kg for diets composed of whole cottonseed [21]. However, the relevance of gossypol studies to commercial cattle operations needs to be carefully considered. The free gossypol content in the cottonseed meal study rations cited above, were obtained from solvent extraction methods, which accounts for less than 2 per cent of the oil extraction method used today. In addition, males in gossypol related studies have not been subjected to actual fertility trials.

Reproductive Management of Cows In herds that use only NS the advantages of a fresh herd and a voluntary waiting period of 60 days used in AI herds should be considered. Fresh cows can be monitored daily for postpartum complications and sick cows treated promptly without the nuisance of having a bull present. Concerns from practitioners that work in herds that use only NS bulls is that the opportunity to observe cows daily for sickness is lost because of the reluctance by herd personnel to enter a pen with a bull in it. The benefits of a well balanced postpartum transition diet in order to reduce metabolic or digestive disorders is also warranted. Further, the use a prostaglandin regiment to promote

multiple cycle and uterine involution in an attempt to increase pregnancy rate at first service can be advantageous . To help reduce the interval from calving to first service cows can be treated with prostaglandin prior to being exposed to the bull. Pregnancy diagnosis can be performed in cows 40 to 60 days after bull exposure. Cows that are found open on examination can be re-examined 30 to 60 days later. Cows that are found to be cystic can be treated with GnRH and use of prostaglandin should be limited to cows with pyometra only. Because of the inaccuracy of breeding dates in many NS herds, it is necessary to accurately estimate gestation length in order to allow the cow an appropriate dry period. The author has found it to be beneficial to re-examine pregnant cows again at 60 to 90 days. In herds in which trichomonosis has been diagnosed, the incidence of early abortions at 30 to 75 days, has been reported to be between 10 and 30 percent. In addition, the diagnosis of pyometra related to trichomonosis is more likely at this time. This will help diagnose this problem early and minimize the economic loss due to this venereal disease. Pregnant cows should also be recomfirmed prior to dry off similar to the practice used in A.I. herd. In most herds that use both NS and AI, cows are turned with the bull after they are found open at a predetermined day in milk (130 to 150 days) regardless of service number. These cows can then be palpated for pregnancy as previously described after they are turned in with the bull.

Conclusion Despite the tremendous evidence supporting the economical advantage of AI compared with NS bull, many dairy producers consider that the use of natural service is advantageous to their reproductive management. Considering the prevailing heat detection and pregnancy rates on a dairy farm, the use of NS becomes a valid option when the effective pregnancy rate (heat detection rate x conception rate) falls below 25 per cent. This option will only be maximized if bulls that are able to impregnate an estrous cow are used. Therefore, bulls should pass a BSE prior to use and should be repeated frequently. With the exception of brucellosis, bulls should undergo the same herd health procedures as the cows. Particular attention should be made to the prevention of vibriosis and trichomoniasis. Reproductive performance monitoring of NS bulls should be conducted on a periodic basis. Attention should be given to the recommended indices for monitoring NS bull performance. Veterinarians should be vigilant with dietary and managerial factors that may impair bull fertility. A tremendous amount of time is spent by dairy consultants in convincing producers not to use NS bulls in their operations. As is often the case, these producers have made certain financial considerations that are unknown by the consultant in order to arrive at the decision to use a bull. Nevertheless, because of the danger involved in using bulls, the possibility of introducing venereal diseases and the loss in genetic progress, use of NS service should be recommended only after all methods to improve pregnancy rate with AI have been exhausted.

Management and Evaluation Considerations for Range Beef Bulls R. Christmas

Elanco Animal Health, Blue Springs, MO, USA.

Introduction The success of most, if not all, cow-calf enterprises depends on the satisfactory reproductive performance of the bull. According to a recent survey 91.9% of cow-calf operations utilize live-cover breeding exclusively [1]. Traditionally, a bull has been considered fertile if he successfully impregnated cows. Successful impregnation of cows will of course remain a minimum requirement. The goals for reproductive performance of an individual operation will vary. With proper management it is not unrealistic to expect a 95% conception rate with 75% of the calves born in the first 21 days of the calving season. In order to achieve these it is essential that herd sires are managed properly. We will focus on developmental influences on bull fertility, key management principles of range bulls and how to identify and maintain fertility in the bull battery.

Fertility Assessment Currently, the commonly used fertility assessment techniques are quite subjective and error prone [2,3]. The unreliability of these techniques may be the reason why less than 3% of cow producers routinely have bulls fertility evaluated [1]. New technologies such as the assessment of heparin-binding proteins in semen may provide more objective data to assess fertility prior to the onset of the breeding season [4]. Currently a breeding soundness examination conducted by the guidelines set forth by the Society of Theriogenology is the most practical and reliable method of determining bulls' functional reproductive capacity prior to the breeding season [5]. The breeding soundness exam should be complete, and assess not just reproductive and seminal characteristics but also a complete assessment of physical parameters. Abnormalities are commonly found with the eyes, feet, and legs. One should watch for chronic ailments (such as lumpy jaw and ocular squamous cell carcinoma). The examination should be timed approximately 60 days prior to the breeding season. By performing the exam early, there is time to correct any problems noted and purchase bulls to replace those with irreparable conditions. The cost of these exams varies from $15.00 to $60.00 us. With an average cow representing a $2,000 dollar investment in land, equipment, facilities and animal unit price and the bull being responsible for approximately 25 cows, it is easy to see that, even despite the inherent subjectivity of these exams, they are still well worth the investment [6]. A number of recent publications from the University of Arizona have shown that heparin-binding proteins on the sperm are positively correlated with fertility [4,7,8]. Heparin-binding proteins are produced by the male accessory glands, secreted into the seminal fluid, and upon ejaculation bind to sperm [9]. In a study conducted at King Ranch, the bulls that passed a standard breeding soundness examination were also tested for seminal heparin-binding protein. They were then placed into groups based on the level of heparin-binding protein in the sperm membrane and seminal fluid. Groups with the largest amount of heparin-binding protein in sperm membranes but not in seminal fluid had greater fertility than did groups with other heparin-binding protein profiles. The heparin-binding protein assay is now commercially available and is being used by cow-calf producers. Additional research needs to be done to conclusively establish if there are genetic, management and or environmental factors that influence the production and distribution of the heparin-binding proteins. Testing bulls for heparin-binding protein may prove to be a valuable selection and management tool in the future.

Nutrition There is an abundance of good research data on how nutrition affects the reproductive performance of heifers and cows. Unfortunately, there is very limited data available on beef bulls. It has been documented that severe dietary restriction results in impairment of fertility. In some cases, the damage has been shown to be permanent [10]. With the current popularity of bull performance testing, it is more often the case that bulls are developed in conditions of dietary energy excess rather than deficiency. It has been shown that excessive dietary energy in developing beef bull rations can have deleterious effects on sperm morphology and motility [11-13]. These effects may be transitory and are thought to be the result of impaired thermoregulation of the testes due to fat deposition in the scrotal region. However, there is a valid concern that bulls developed in this fashion may be predisposed to certain reproductive career-ending musculoskeltal disorders such as osteochondrosis dessicans and laminitis [14]. Ideally bulls should be in a body condition score of 6 to 7 prior to the onset of the breeding season. Often young bulls purchased at sales are in excessive body condition. If this is the case it has been recommended that young bulls carrying excess flesh should be "let down" from the time of purchase until they are turned out with the cow herd [15]. It should be stressed that both over-fat and over-thin bulls may have reduced libido and fertility. During the breeding season, old and/or thin bulls may benefit from supplementation through hand feeding of small amounts of grain, given daily or every other day [16].

Bull to Female Ratio The standard recommendation for a bull-to-cow ratio is 1:25 and this is typically adequate although it makes allowance for sub-standard bulls. Research has shown that this ratio can under-utilize bull power [17,18]. Under-utilization of bull power with a 1:25 ration is especially true in situations where the stocking density is high due to an abundance of high quality native forage, and when the bull is highly fertile. Libido and serving capacity of bulls are helpful components to assess in order to optimize the bull to female ratio [19].

Vaccination Health care management for breeding age bulls varies according to their age. To facilitate the delivery of a well designed health program it is helpful to manage the bulls in age groups. The suggested groups would be yearling to two-year-olds and mature bulls. Each group has its own unique health concerns. The program should be targeted at disease prevention and reproductive development and efficiency [20]. Vaccination programs for the two groups are similar. In the yearling bull the focus is establishing a long-term immune base for the common viral and reproductive diseases. For the viral diseases IBR and BVD, modified live vaccines (MLV) have been shown to provide long term immunity, up to five years. However, MLV IBR vaccination may compromise the export of frozen semen. Initial vaccination for campylobacterosis and leptospirosis should also be administered. Timing of the vaccination is very important. Usually, it is best to administer vaccines to yearlings at the time of semen evaluation and booster two to three weeks prior to the onset of breeding season. Mature bulls should not require a booster and can be vaccinated at either of the two previously mentioned times. Other vaccines that maybe considered are anaplasmosis, trichomoniasis, and any other disease that maybe endemic for a particular area. In order for vaccinations to be effective a sound management and nutrition program must be in place.

Parasite Control Parasite control will vary from region to region. Generally, bulls will require deworming once yearly. However, in climates such as that in the Southeastern United States, it may be necessary to deworm bulls two to three times per year. Fly control is an essential part of any bull management program. Fly strike in the scrotal region can lead to enough heat and inflammation that thermoregulation of testes is altered and fertility is subsequently impaired.

Observation during the Breeding Season It is essential that bulls be observed closely during the breeding season. The producer should observe that the bull not only mounts the cow but also that copulation is taking place. Observation is of particular importance in the first several days of the breeding season in multiple sire pastures because injuries are common as dominance is being established. Range bulls are prone to back, feet and leg injuries during the breeding season. Bulls may also have injuries to reproductive structures. Common reproductive injuries include preputial lacerations and penile hematomas (broken penis). Any of the previous conditions can resulte in reduced pregnancy rates and an altered pattern of conception. If a bull fails to breed a cow early in the breeding season this will result in the cow remaining open until at least the next estrous cycle resulting in a minimum 45 to 50 pound reduction in weaning weight [20].

Culling Age is a major consideration when making culling decisions for beef bulls. Semen quality begins to decline after age 6 and this also about the same age when mature bulls lose their social dominance rank to younger more aggressive bulls. If a bull over age 7 has exceptional value it is recommended to utilize that bull in single-sire pastures, or by hand mating, and ensure that he passes a thorough breeding soundness exam [21]. Other factors that should be considered when making culling decisions are vision, conformation and disposition. Also bulls that produce poor performing calves should be ear marked for culling.

Summary Listed below are key management techniques recommended to obtain optimum fertility from range beef bulls [21].

• Control disease with appropriate vaccinations. • Provide a well-balanced nutritional program year round. • An annual breeding soundness exam six to eight weeks prior to the onset of the breeding season. • Observe bulls throughout the breeding season for their ability to mate. • Use separate pastures for bulls less than four years of age and greater than six years of age to avoid

dominance problems. • Cull bulls with poor vision, low semen quality, lack of desirable conformation and those producing inferior

calves.

Bull Sex Drive and Reproductive Behavior P. J. Chenoweth

Large Animal Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

Introduction Although they are not generally considered to be seasonal breeders, cattle are subject to seasonal influences upon reproduction [1] which may be associated with ambient temperature, feed availability and parasite loads. Evolutionary adaptation to regional environments often resulted in a consistent pattern of a predominantly Spring calving pattern, with mating occurring in the early summer [2]. Prehistoric cattle are assumed to have formed relatively small matriarchal groups on local range, which bulls inhabited during the period of breeding opportunity. Here, dominant males derived pre-eminent access to receptive females. Cattle are polygynous; a system which allows individual males to mate with multiple females [3]. In general, beef cattle systems throughout the world are still heavily reliant upon natural breeding [4,5]. Modern production systems, whether employing natural or artificial breeding, have challenged many evolutionary adaptations, by altering environments, changing social groupings, and by reducing both breeding "season" length and male to female ratios. This places greater emphasis on those factors important for male reproductive success, including male sex-drive, or libido [6].

Definitions Sexual behavior in the bull includes the detection, courtship and service of estrous females. Libido, or sex drive, has been defined as the "willingness and eagerness" of a bull to attempt mount and service of a female [7], whereas mating ability describes his physical ability to complete service. Serving capacity is a measure of the number of services achieved by a bull under stipulated conditions [8] and thus includes aspects of both libido and mating ability. Reaction time is that time which elapses between male cognition of an appropriate stimulus and the completion of service [7].

Cattle Reproductive Behavior At pasture or range, bulls are initially attracted towards females by the sight of mounting activity. The major incentive to attempt mount or service is an immobile female or similar inverted U structure. Where this exists, the bull will often attempt service regardless of the estrous status of the female; in fact, steers are successfully employed as mount animals in semen collection centers. Pheromones also play a role in allowing bulls to detect receptive females, although this mechanism evidently requires close physical contact for activation in cattle [9]. The major special sense used by bulls to detect estrous females is vision [10,11]. The task of identifying estrous females is facilitated by the tendency of females in both late pro-estrus and estrus to form a mobile sexually-active group (SAG) which usually stays within visual contact of the bull or bull group [7,12]. Females in heat generally become more active and vocal than at other phases of the estrus cycle. Bulls tend to be most attracted to females newly in estrus and provision of a fresh stimulus female can restore libido in satiated males [13,14]. Bulls test the receptivity of females by making real or sham mounting attempts, by chin resting and by licking and sniffing around the perineal region. The last actions are often closely followed by a characteristic curling of the upper lip, termed flehmen. This action is most probably associated with fluid transfer to the vomeronasal organ where it is assessed for pheromonal activity [9]. Females can exert considerable control over mating by determining the timing of sexual access, and discriminating between competing males. Bulls form hierarchal groups, with social status influencing reproductive success [7,14]. Prestimulation of males increases their sexual response [15]. Bulls possess a fibro-elastic penis and copulation generally occurs rapidly (1-2 seconds) once intromission is achieved [16]. Bulls are capable of short bursts of great reproductive activity, dependent upon their inherent sex drive and stimulus pressure. Breeding trials with both natural and induced-estrus females indicate that it is not uncommon for bulls to serve multiple times (20 - 30 plus) within a 24 hour period.

Biostimulation Male animals are capable of triggering neuroendocrine reflexes which alter ovarian function in conspecific females [17]. Where stimulatory, such effects have been collectively termed biostimulation [18]. In various species, biostimulation has been shown to advance onset of puberty, initiate estrus post-partum and alter temporal relationships associated with estrus and ovulation. In cattle, biostimulatory effects do occur, although they are less dramatic compared with sheep and swine. There are indications that biostimulation is advantageous in reducing postpartum interval in cows, although nutritional interactions occur [19]. Androgenized females can be as effective as biostimulators as can bulls. Less evidence is available in support of positive biostimulatory effects on the advancement of puberty in heifers where the appropriate nutritional and social interactions remain to be adequately defined [19,20].

Tests for Bull Sex Drive Libido, or sex-drive, is a measurable behavioral trait. Testing procedures for bulls generally rely on the exploitation of several or more of the following findings:

1. Libido in bulls has a large genetic component [21-26]. 2. Bulls are polygamous and tend to distribute their services among receptive females [8]. 3. The greatest single stimulus for a bull to attempt mount and service is the immobile rear end of a female,

or something he perceives as similar [27-29]. 4. Prestimulation of bulls increases their sexual response [15,28] 5. Competition among bulls can increase their sexual response [4,30].

Many attempts have been made to assess sex-drive in bulls and other male livestock [31]. As simple observation of male sexual activity in the breeding pasture has generally yielded disappointing quantitative results, a number of researchers have devised formalized, pen-type testing regimes. Early studies on bull sexual behavior sought to counter difficulties in maintaining sex drive in dairy bulls in AI centers [32], and subsequently, to determine the role of sexual behavior in optimizing sperm harvests [13,33]. In Sweden, a "libido index" used to assess both libido and mating ability in A.I. sires [22] was subsequently modified ("libido score") for assessing range-type beef bulls [7,34]. A "serving capacity" test was developed in which bulls are group-tested with restrained, non-estrus females at BFRs of either 5:2 or 5:3 for 40 to 60 minutes [8] and scored on the basis of services performed during the observation period. Comparison of reaction time, serving capacity and libido scores in young Bos taurus bulls [27] indicated the following; 1) that libido score was most repeatable, restrained females were equally attractive to bulls whether they were in induced estrus or not, and a 10 minute test provided as much comparative information on bull sex drive as did a 30 minute test. The use of estrous females was shown to be unnecessary for the assessment of sex drive in Bos indicus bulls also, providing females are adequately restrained [35], although one study showed that estrous status (as well as cow identity) influenced service rates in Santa Gertrudis bulls [36]. Testing procedures for bulls have been developed which exploit elements of both the libido and serving capacity score systems [31,37-39].

Test Predictability and Repeatability The ideal sex drive assessment for bulls should be simple, quick, highly repeatable, predictive of reproductive performance and esthetically acceptable. At present, no single current procedure fulfils all of these criteria. However, it is possible to reliably estimate relative differences in sex drive between bulls [14]. For example, moderate phenotypic correlations (r = 0.67 and 0.60 respectively) were obtained between libido and serving capacity scores in yearling bulls tested on different days [27], although reaction times to service in the different tests were not significantly correlated. Lack of predictability for reaction time was confirmed in a subsequent study with young Hereford and Angus bulls [40]. With Bos indicus bulls, the repeatability of libido scores was relatively low (r = 0.44) [41], although this procedure was regarded as being superior in repeatability and logistics to the serving capacity method for assessing sex drive in mature Bos indicus bulls [35]. When 26 yearling Bos taurus bulls were assessed a total of 8 times (two tests per day on four occasions over a 9 week period) for libido and serving capacity scores, four or more tests were required to significantly reduce test variance [4], although bulls which scored highly in the first test period generally scored highly in subsequent tests. Bulls which achieved low scores at the first test period either improved dramatically at some point in the testing regime, or else remained at a low level throughout. For the former group, a maturing and/or learning process was evident - a process which can adversely affect test results in young, inexperienced bulls. This phenomenon has been observed in other trials [4, 27,37] where young bulls obtained low serving capacity scores which improved with subsequent mating experience. Young, virgin bulls which achieve poor serving capacity results should be retested after they obtained sexual experience [37]. This resulted in improved scores in young virgin Santa Gertrudis bulls [42]; a group which has been difficult to adequately pen-test [43]. Best results have generally been obtained when assessments of libido or serving capacity are used to rank bulls or place them into categories or groups (with high scoring bulls being most predictable). Thus it was found that eight Hereford bulls maintained their relative ranking for both libido scores and fertility when assessed at both 16 and 40 months of age [44]. In another study [28], high correlations (r = 0.82 to 0.91) were obtained for rankings in mating activity between simulated pasture tests and subsequent pen tests of 12 Bos taurus bulls.

Fertility Relationships Cattle fertility is a multi-factorial trait with involvement of both male and female factors. From the male aspect, there is good evidence that bull libido is one of the more important contributing factors. For example, in one study, better first-cycle pregnancy rates were obtained in heifers mated with higher serving capacity bulls than in those mated with bulls of low serving capacity [45]. More recently, differences in pregnancy rates were demonstrated between high, medium and low serving capacity Hereford bulls [46]. Other studies have shown advantages in herd fertility for higher sex drive bulls [47-50]. Close relationships were reported between bull rankings for fertility, libido score and testosterone response to parenteral GnRH challenge [51]. In a Florida study, bull libido and

semen quality both significantly influenced pregnancy rates achieved by naturally mated Brangus bulls, with libido having most effect (A.C. Warnick, personal communication), while a study with Bos indicus bulls in Mexico provided positive relationships between tests for bull sex-drive (libido and serving capacity scores) and reproductive performance [52]. Other studies have indicated either that bull libido assessment provided greater prediction of bull fertility than did semen assessment alone [50], or that it augmented traditional breeding soundness evaluation [53]. Using multi-sire mating and progeny identification by blood typing, the number of services performed in libido/serving capacity tests was positively correlated with fertility up to a level of approximately four services [54]. Above this level of services, however, fertility appeared to decline. Despite such reports, however, others have shown either poor or inconclusive relationships between bull libido/serving capacity assessment and either herd fertility or bull performance at pasture [37,41,55-59]. In some studies, although higher libido bulls serviced more often, and serviced more females, than did lower libido bulls, more pregnancies did not result [4,37,58]. In northern Australia, prior assessment of bull sex-drive was generally not predictive of bull performance in multi-sire breeding trials [42]. A number of reasons may occur for such apparently contradictory findings, including differing approaches and methodologies. For example, bulls may not have been placed under sufficient breeding stress to demonstrate real differences. Such differences may have become apparent with use of higher BFRs or shorter breeding periods. Social interactions between bulls may mask differences in reproductive potential. In addition, in cooperator breeding trials, bulls of potentially low fertility are often excluded. A major potential problem exists whenever investigators attempt to demonstrate that a single trait (e.g., bull sex drive) has a consistent, decisive influence on herd fertility. This is because cattle fertility is a factorial, influenced by both male and female factors. Male factors include sex drive, mating ability, sperm numbers and semen quality. Scrotal circumference, sperm motility and morphology can separately influence fertility [53], and these are apparently not linked with sex drive in bulls [23,37,49,60,61]. Bulls may be superior in one trait, or several, but their fertility may be compromised by deficiencies in others. This was demonstrated in one study [58] where 92 beef bulls were placed both into satisfactory and questionable BSE categories, as well as into high (score 9 to 10) and medium (score 7 to 8) libido categories prior to single-sire mating with groups of estrus synchronized heifers. Here, pregnancy rate was 9.1 percent higher for satisfactory BSE bulls compared with those in the questionable BSE category. However, pregnancy rate did not differ between bulls of high and medium libido score even though high libido bulls serviced more females and served more times than did medium libido bulls. This paradox apparently occurred because a lower percentage of serviced females in the high libido group became pregnant than in the medium libido group. Here, differences between bulls in sex drive were masked by differences in BSE traits. The ability of bulls to service females is related not only to their inherent sex drive but also to their mating ability. Problems in mating ability may be due to a number of physical and pathological causes including skeletal and penile abnormalities [18].

Factors Influencing Bull Sex Drive 1. Age, Rearing and Nutritional Effects - Age and (or) experience of bulls can influence their mating ability and thus their apparent sex- drive. Competent mating ability does appear to have a learning component in bulls [26,37,62-64] , even though exposing young Polled Hereford bulls to heifers post-weaning did not influence subsequent libido or mating ability [65]. In this trial, individually-penned bulls initially showed greater serving capacity than did group-penned bulls, but this difference did not persist. Male-male mounting in group penned bulls was not indicative of libido, serving capacity or mating behavior with heifers. It was concluded that social restriction of young bulls was not detrimental to their mating ability. In young tropical beef bulls, libido score increased with bull age between 16 and 31 months of age [38]. Bull age affected sexual behavior traits in crossbred bulls, with yearling bulls showing lower libido and a higher proportion of mounts than older bulls [54]. In Florida, sexual performance assessments generally increased with age in young (12 - 24 month) Bos taurus bulls, although not in Bos indicus bulls [66] which generally displayed a lower level of sexual activity. More information is needed to differentiate the effects of age and experience on bull sexual behavior from those due to environmental and managerial influences. Zebu bulls raised on open range exhibited slower sexual responses compared with those reared more intensively [67], although no permanent sexual inhibitions are attributable to rearing methods in bulls [48]. It is, however, possible that temporary sexual inhibitions in bulls may compromise pregnancy rates in herds which have restricted breeding seasons [7]. Nutritional effects on bull sex-drive have generally not been well characterized [21,68-71]. Prolonged nursing was considered to retard, or compromise, the expression of normal sexual behavior in Angus bulls [72] as was feeding high concentrate levels to crossbred bulls [69]. However, postweaning dietary energy levels were not found to affect sex drive in young bulls of synthetic breeds [53]. Negative relationships were obtained between sex-drive and production traits (average daily gain (ADG) and final test weight) in yearling beef bulls in one study [25], whereas underfeeding had no adverse effects on bull sexual behavior in another [70]. Indirect effects of overfeeding might include obesity as well as feet and leg problems; all of which could contribute to lowered sex-drive [73].

2. Bull to Female Ratio (BFR) - Traditional recommendations for bull-to-female ratios (BFRs) in natural breeding herds of 1:20 - 1:30 can underestimate the reproductive capabilities of competent bulls. For example, in a study in

which single- and multi-sire breeding systems were compared using Hereford bulls at BFRs of 1:25, 1:44 and 1:60 [74], fertility, libido and mating ability of individual sires was more important than either BFR or single- vs multi-sire breeding systems. Similarly, in northern Australia, there was no difference in reproductive rates between single- and multi-sire herds studied over a period of 18 years [75]. In Colorado, yearling Hereford bulls which had been pre-assessed for BSE and libido, were compared at BFRs of 1:20 and 2:40 with estrus-synchronized crossbred heifers [76]. Overall, bull mating performance and pregnancy rates did not differ between BFRs. Comparison of a variety of single-sire BFRs (1:7 to 1:51), also with estrus-synchronized females, found that BFR was not a limiting factor to fertility, even at the lowest bull ratios [64], although another study suggested that lower fertility may occur when BFRs exceed approximately 1:50 [77]. In northern Australia, no difference was observed in herd fertility when reproductively sound Brahman bulls were used at BFRs of either 1:17 or 1:40 [78]. Two studies conducted in different environments showed that BSE-screened bulls increased herd pregnancy rates at reduced BFRs (1:20 to 1:33) [79,80]. Thus it is apparent that many bulls can handle considerably more females in a generic breeding season than traditional recommendations would suggest. It is also evident that most producers have yet to take full advantage of these findings. For example, USA surveys indicate that cow-calf operations overall use yearling bulls at 1:17.5 and mature bulls at 1:25; figures which have changed little in recent years (USDA-NAHMS 1998). Cow-calf producers in the Rocky Mountain West used a mean BFR of 1:21, with 25% of herds employing a BFR of <1:18 [81]. Several studies indicate that BFR is relatively unimportant when conducting tests for sex drive in young, experienced bulls [39,49] provided group sizes are relatively small. One study obtained better results when bulls were tested individually [65] although inter-male aggression and interference in group tests may have influenced these results. Such interference can occur more often when there is inadequate spacing between stimulus females, when older bulls are being tested or when bulls are tested in groups greater than 2 [27].

3. Social Effects - Social ranking of bulls within groups can influence their sexual activity [7,74,82,83]. Dominance is expressed more strongly and linearly in older bulls (i.e. those 3.5 to 4 years of age or older) and it appears to be more related to seniority than to either age or body weight [82]. It has been suggested that the effect of social interactions among bulls on herd fertility may be greater at lower BFRs than where there are higher levels of breeding stress [82]. Dominance effects among bulls can also influence results obtained in pen tests for bull sex drive [84]. Dominance rank was negatively correlated with sex-drive in one study with yearling bulls [25]. If dominance and sex-drive do indeed represent different traits, then the dominant bull (or bulls) could impair herd fertility both through failure to service females and also by preventing less dominant bulls from serving. Evidence exists for such effects in extensive beef operations [80], where it was also shown that the social dominance ratio of bulls had some relationship with herd fertility. Such effects are probably most evident when older and younger males are combined in the breeding pasture [82], although mixing different bull genotypes in the breeding pasture may apparently cause similar effects [85]. Social effects may, however, also be beneficial for bull sexual behavior. Such an effect was observed in one study where greater sexual activity occurred when young, inexperienced bulls were tested in groups [3-5] than when tested individually [48]. Similarly, yearling bulls of higher serving capacity achieved more services in double-sire tests than when tested alone [4]. The combination of bull prestimulation and competition can positively influence results of sex-drive test procedures [31].

4. Genetic Effects - The evidence for genetic influences upon bull sex-drive is strong. In Scandanavia, monozygous twin bulls raised on differing nutritional regimes displayed greater similarity within pairs in mating behavior and temperament than between pairs [21], suggesting strong genetic influences upon these traits. Paternal half-sibs of Swedish bulls differed significantly in libido with greater variation between sire-son groups than within them [22]. Studies showing that cross-bred bulls generally exhibited higher sex-drive in pen-tests than did their parental pure breds, indicate that genetic effects, in this case heterosis, influence bull sex-drive [38,86]. In Colorado, line of breeding (inbred lines or crosses among inbred lines) was an important source of variation in bull sex-drive, indicating that lines had previously been selected or differentiated on the basis of sexual behavior [23]. Similarly, differences in libido scores were observed between breeding lines and sires-within-lines in young bulls of British breeds [25]. Here, high sex-drive was not synonymous with either superior production traits (average daily gain or final test weight) or high social ranking. Sire strongly influenced serving capacity in young Angus bulls [26]. A number of studies have indicated that measures of traditional breeding soundness criteria, such as scrotal circumference and semen traits, are not significantly correlated with sex-drive estimates in bulls [23,24,37,49] indicating that these are separate traits. An heritability estimate of 0.59+0.16 was obtained for serving capacity in a study of 157 paternal half-sib bull groups in Australia [24]. In this study, the inclusion of bodyweight as a covariate did not alter the result, and serving capacity was not associated with temperament rating. Breed effects have also been noted in bull sexual behaviour. Bulls of dairy breeds are reputedly more sexually active than those of beef breeds [33] and Bos indicus bulls often display lower, and more variable, levels of libido than do Bos taurus bulls [43,66,86]. In several studies in tropical Australia, Brahman and Brahman crossbred bulls obtained the lowest libido scores, Africander bulls and their crosses achieved the highest, whereas

European bred bulls were intermediate [38,86]. In the US, Bos taurus bulls also obtained higher sex drive scores than did Bos indicus bulls [66]. Despite such findings, a comparison of trials in which bulls were placed with estrous synchronized females indicated that Bos indicus derived bulls were as efficient as European breed bulls in detecting, serving and impregnating estrous females, despite a lower service rate [6]. This discrepancy may be part explained by perceptions that Bos indicus bulls tend to be selective and shy breeders, and that they generally do not perform well in pen tests to assess sex-drive [42,66] , even though they can be very active and efficient in detecting of estrous females in the pasture. In Florida, Bos taurus (Angus, Hereford) bulls obtained the highest scores in sex-drive tests than did tropicalized Bos taurus (Senepol, Romosinuano) bulls, with Bos indicus (Brahman, Nellore x Brahman) bulls generally obtaining lowest scores [66]. Here it was suggested that commonly used testing procedures for sex drive may disadvantage Bos indicus bulls. Suggested test modifications for such bulls include use of unrestrained estrous females, and the minimization of distractions during the test [85], although use of restrained estrous females did not provide an advantage in one trial [42].

Alternative Assessments The indirect determination of bull libido, e.g. via blood hormone levels, is an attractive proposition as it has the potential to reduce or eliminate the time, labor, esthetic or welfare concerns which might occur with current methods of assessing sex-drive. It would also allow assessment of bulls which do not respond well to such testing procedures. However, attempts to link either sporadic or sequential luteinizing hormone (LH) or testosterone (T) levels with bull sex-drive have generally been disappointing [7,27,88] probably because of the episodic nature of hormone release and the inhibiting effects of handling or restraint of the animal. By inducing LH or T release with parenteral administration of gonadotropin releasing hormone (GnRH), some of these difficulties may be circumvented. In one study [51], a significant relationship occurred between induced T levels and bull fertility, while in another [89], positive relationships were obtained with induced LH levels. However, other studies have obtained disappointing results when attempting to relate GnRH induced levels of testosterone or LH with bull sex drive [71,90].