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Animal Reproduction Science 123 (2011) 180–186

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

ong term effect of Ovum Pick-up in buffalo species

ianluca Negliaa,∗, Bianca Gasparrinia, Domenico Vecchioa, Lucia Bocciaa,ttore Varricchiob, Rossella Di Paloa, Luigi Zicarelli a, Giuseppe Campanilea

DISCIZIA, Faculty of Veterinary Medicine, “Federico II” University, Naples, ItalyDepartment of Biological and Environmental Science, Sannio University, Benevento, Italy

r t i c l e i n f o

rticle history:eceived 27 May 2010eceived in revised form0 December 2010ccepted 10 January 2011vailable online 20 January 2011

eywords:vum Pick-up

n vitro embryo production (IVEP)uffaloumulus oocyte complexes (COCs)

a b s t r a c t

The aim of this study was to evaluate the effect of an Ovum Pick-up (OPU) treatment carriedout for 9 months in buffalo (Bubalus bubalis) species. Eight pluriparous non-lactating buffalocows underwent OPU for 9 months. Recovered cumulus enclosed oocytes (COCs) were clas-sified and COCs suitable for in vitro embryo production (IVEP) were in vitro matured (IVM),fertilized (IVF) and cultured (IVC) to the blastocyst (Bl) stage. Animals were monitored for atotal period of 270 days, but at the summer solstice, follicular turnover decreased and at the68-day of the trial, we decided to increase the OPU sampling interval from 3–4 to 7 days. Itwas therefore possible to distinguish two phases: a first phase (18 sessions), during whichOPU was carried out twice weekly and a second phase (16 sessions) during which OPU ses-sions were performed weekly. This reduction did not modify the percentage of good qualityCOCs, while the incidence of grade D COCs decreased (P < 0.01). Furthermore, embryo pro-duction was higher in the second phase, either if embryos were calculated on the totalrecovered COCs (8.3% vs. 21.4%; P < 0.01) and on grade A + B COCs (13.0% vs. 32.1%; P < 0.01),that supposedly should have given similar blastocyst yield. During the total period of thetrial it was possible to distinguish a first period of 6 months (34 sessions) characterizedby blastocyst production (0.36 blastocyst/buffalo/session), followed by an unproductiveperiod of 3 months (12 sessions), during which embryos were not produced. During thefirst 6 months a higher (P < 0.01) number of follicles (5.06 vs. 3.71), small follicles (3.38vs. 2.07), total COCs (2.58 vs. 1.56) and good quality (A + B) COCs (1.51 vs. 0.94) per sub-ject/session were recorded compared to the last 3 months. No Blastocyst were produced

during the second period, even if the percentage of grade A + B COCs was similar to thatrecorded during the first period. In conclusion, buffalo cows submitted to repeated OPUsampling for a 9-month period, showed a decline of follicle recruitment and oocyte col-lection after the first two months of samplings. After 6-month of samplings, in spite of thequality grade of the collected oocytes, we found a drop in their developmental competence.

∗ Corresponding author at: DISCIZIA, Department of Animal Sciencend Food Inspection, University of Naples, V. Delpino 1, 80137 Naples,taly. Tel.: +39 081 2536063; fax: +39 081 292981.

E-mail address: neglia@unina.it (G. Neglia).

378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2011.01.011

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The application of Ovum Pick-up (OPU) technology

(Pieterse et al., 1988; Boni et al., 1993) is currently car-ried out in donors of different ages, from 2 months oldcalves (Armstrong et al., 1992) to elderly subjects, as wellas in different physiological conditions (Galli et al., 2001).In fact, OPU does not interfere with the physiological sta-

duction

G. Neglia et al. / Animal Repro

tus of the donor and is simply feasible and repeatable (Boniet al., 1997a). Furthermore, it allows the visualization andthe aspiration of all follicles with diameter >2 mm, whichresets the estrous cycle and avoids the dominance of onefollicle over the subordinates. It has previously been shownin several species that the optimal interval between aspi-ration sessions is 3–4 days (Galli et al., 2001; Boni, 1997).Therefore, with this approach, it is hypothetically possibleto avoid the atresia of subordinate follicles and the follicu-lar waves increase from 2–3 for each cycle to 6 in the sameperiod. This results in the production of a higher numberof cumulus oocyte complexes (COCs) usable for in vitroembryo production (IVEP), as well as with an improvedoocyte quality (Pieterse et al., 1991; Kruip et al., 1994; Galliet al., 2001).

In cattle, this technique is applicable for long period,without interfering with the reproductive status of thedonors (Pieterse et al., 1991; Galli et al., 2001). Indeed, ithas been shown that the extension of OPU to 6 monthsdoes not affect neither oocyte characteristics nor the IVEPefficiency (Kruip et al., 1994; Boni, 1997). On the contrary,the number of follicles progressively increased until thethird month, and then subsequently decrease (Boni et al.,1997a).

The low response to superovulation treatmentsobserved in buffalo species (less than 2 embryos/animal;Zicarelli, 1997b) has increased the interest of severalscientists in IVEP, especially if a non-invasive procedurefor recovering oocytes is used. OPU has been successfullyapplied in buffalos even if the efficiency is lower in termsof punctured follicles and oocytes suitable for IVEP (Boniet al., 1996, 1997a,b; Boni, 1997; Neglia et al., 2003).The technique was used for the first time on deep anoe-strous buffaloes under ovarian hypotrophic conditions(Boni et al., 1996). Subsequently, it was performed indonors of different ages, reproductive status and withor without hormonal treatments. When a single OPUsampling was carried out from the 6th to the 11th dayof the estrous cycle in buffalo cows with less than 150days open, a large follicular population was observed(Boni et al., 1995). After that it was also demonstratedthat the increase of days open negatively influences thefollicular population (Boni et al., 1997b). The applicationof OPU in prepubertal buffalo calves (Techakumphu et al.,2004; Presicce et al., 2002) that received hormonal treat-ments supplied a changeable number of follicles, varyingbetween 21.3 (Presicce et al., 2002) and 6.6 (Techakumphuet al., 2004). Hormonal treatments increase the numberof recruited follicles and recovered oocytes also in cyclingand lactating buffaloes (Promdireg et al., 2005), whileno experiences have been performed in non lactatingbuffalo cows.

Considering the low number of recovered oocytes inbuffalo, the possibility to carry out OPU for long period oftime, is of a great importance to exploit the germplasmof the best donors. With this approach, it would be in

fact possible to rescue the genetic material of highlyproducing cows at the end of their productive and repro-ductive career, before they are slaughtered. These subjectsmay represent an important genetic source for buffalospecies. However, in this species OPU has never been car-

Science 123 (2011) 180–186 181

ried out for a longer period than 3–4 months (Campanileet al., 1999; Manjunatha et al., 2008a,b). Furthermore, theonly study performed in buffaloes at more than 500 daysopen (Boni et al., 1996) was carried out for four months.Since buffalo is a short-day breeder (Zicarelli, 1997a), itis not possible to rule out that the season may be alimiting factor in extending the period of oocyte collec-tion/donor.

Therefore, the aim of this study was to evaluate theeffect of an OPU treatment prolonged for 9 months on non-lactating Italian Mediterranean Buffalo cows at the end oftheir productive career.

2. Materials and methods

All chemicals and reagents, if not otherwise stated, werepurchased from Sigma (Sigma–Aldrich, Milan, Italy).

2.1. Ovum Pick-up and COCs processing

The trial was carried out on 8 pluriparous non-lactatingItalian Mediterranean buffalo cows at the end of theirproductive career which were located in a commercial buf-falo farm in the Campania region. Before the start of theexperimental period, buffaloes were examined by ultra-sonography and two 5 cm3 injections of PGF2� analogue(Dinolytic, Pfizer Animal Health Srl, Italy) were adminis-tered 12 days apart only to the subjects that did not showany genital tract abnormality. Three days after the lastinjection of PGF2� the estrus was checked by rectal pal-pation and ultrasonography: the animals that showed afollicle larger than 1 cm and a tonic uterus with or with-out mucus vaginal discharge were included in the study.OPU started on day 3 after estrus. The animals underwentOPU for a total period of 9 months. The donors were con-tained in a chute and an epidural anesthesia (4 ml of 2%lidocaine hydrochloride – Gellini, Italy) was administeredbefore OPU.

Ovum Pick-up was carried out by the same opera-tor as previously described (Neglia et al., 2003). Briefly,the OPU setting consisted of a portable ultrasound unit(Aloka SSD-500, Aloka Co., Tokyo) with a 5 MHz sectorscanner and a metal guide to fit 17 gauge needles bothallocated in a properly designed vaginal guide. A vacuumpressure of −40 mm Hg was constantly maintained usinga regulated vacuum pump (K-MAR-5100, Cook IVF Co.,Australia) and the aspiration line was continuously rinsedwith 25 mM hepes-buffered TCM 199 supplemented with100 USP units ml−1 of heparin and 10% fetal calf serum(FCS) during follicular aspiration. The tubes for oocytes col-lections were constantly maintained at 37 ◦C. All visibleantral follicles were punctured and classified into three cat-egories, according to their size: small (diameter < 0.5 cm),medium (diameter between 0.5 and 1 cm) and large (diam-eter > 1 cm).

The COCs were searched immediately after follicular

aspiration by filtering the aspirated follicular fluid and aspi-ration medium, and classified into 5 categories: grade A(oocytes with more than three layers of granulosa cellsand homogenous cytoplasm); grade B (oocytes with atleast 2 layers of granulosa cells and homogenous cyto-

1 duction

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82 G. Neglia et al. / Animal Repro

lasm); grade C (oocytes partially or totally denuded);rade D (degenerated oocytes) and grade E (expandedocytes but with homogeneous cytoplasm, typical of thestrous phase). It is worth specifying that grades A andCOCs are considered suitable for in vitro embryo pro-

uction in this species. The recovery rate (calculated ashe percentage of total number of COCs in relation tootal number of follicles) was also recorded for eachonor.

The COCs were washed twice in hepes-buffered TCM99 (H 199) with 10% FCS and then allocated in theame medium supplemented with 50 �M cysteamineGasparrini et al., 2000) and 0.5 �g ml−1 FSH, 5 �g ml−1

H, 1 �g ml−1 17-�-estradiol (Caracciolo di Brienza et al.,001). Processed oocytes were stored in 15 ml Falcon tubes

n a portable incubator at 38.5 ◦C and moved to the labithin 4–6 h for in vitro embryo production (Gasparrini,

002). The COCs recovered from each donor were kept sep-rated throughout the IVEP procedure.

.2. In vitro embryo production (IVEP)

For in vitro maturation (IVM) the COCs of each buffaloow were transferred into 50 �l droplets under medical oilf the final maturation medium, consisting of bicarbonate-uffered TCM 199 (B199) with hormones and cysteamine inhe same concentration previously described. The dropletsere incubated at 38.5 ◦C for 22–24 h under controlled gas

tmosphere of 5% CO2 in humidified air.In vitro fertilization (IVF) was carried out according to

he method previously described by Parrish et al. (1986)he day after IVM. The sperm of one bull, previously testedor IVF in our laboratory, was treated by swim-up pro-edure in a modified version of Ham’s medium for 1 h.he pellet obtained after centrifugation of supernatant wasesuspended to a final concentration of 2 × 106 ml−1 in theertilization medium, a modified TALP supplemented with.2 mM ml−1 penicillamine, 0.1 mM ml−1 hypotaurine and.01 mM ml−1 heparin. Fifty �l fertilizing droplets coveredy mineral oil were incubated under the same gas atmo-phere as for in vitro maturation.

After 20–22 h of co-incubation with spermatozoa, pre-umptive zygotes were cultured (IVC) in 20 �l droplets ofOFaaBSA (Tervit et al., 1972; Gardner et al., 1994) for 7ays in modulation chamber with a gas atmosphere of 5%O2, 7% O2 and 88% N2. On day 5 (day 0 = IVF day) the cleav-ge rate was assessed and the embryos were transferrednto fresh droplets of the same medium for further 2 daysf culture. Final embryo output, in terms of blastocyst (Bl)ield, was evaluated at day 7 of culture.

.3. Statistical analysis

Mean values for all the parameters analyzed in eachhase and period, defined retrospectively on the basis ofhe results, were calculated from the data recorded in all the

nimals in each OPU session. The differences between allhe data expressed in means ± standard error (mean ± SE)ere carried out using ANOVA, utilizing a repeated mea-

ures model (SPSS 17.0, 2009). Percentage data (percentagef different grades of follicles, recovery rate, percentage

Science 123 (2011) 180–186

of different grades of COCs, cleavage rate, blastocyst rate)were analyzed using the Chi-square test.

3. Results

During the trial a significant (P < 0.01) reduction of thefollicle number was observed since the 13th PS (6.2 ± 0.24vs. 4.6 ± 0.28, respectively for 1–12 PS vs. 13–18 PS). Fur-thermore, after 34 sessions, no embryos were producedby in vitro procedure, although COCs were still recovered.Therefore, on the basis of the results obtained in the presentstudy, the trial was divided in two periods: the first, fromMarch 23th to September 18th (179 days; 34 sessions), dur-ing which the buffaloes yielded embryos in vitro and thesecond, from September 21th to December 18th (91 daysand 13 sessions), in which no embryos were produced.

Furthermore, within the first period, it was possible todistinguish two phases: during the first phase, from March23th to May 25th (63 days and 18 sessions), the transvagi-nal ultrasound aspiration was performed twice weekly,while in the second phase, from June 1st to September 18th(116 days and 16 sessions), only once weekly, as a result ofthe reduced follicular turn-over. In the second period OPUwas also performed once weekly until December 18th.

3.1. Differences between I and II phase within the firstperiod

A significant (P < 0.01) reduction of the total numberof follicles was observed in the second vs. the first phase(Table 1). However, both the number and the incidenceof large follicles significantly (P < 0.01) increased, whereasonly the number of medium and small follicles was reduced(P < 0.01) in the second vs. the first phase.

In the second phase the total number of COCsalso decreased (P < 0.05); however, no differences wereobserved in the number and the incidence of grades A, B andC COCs. On the contrary, the incidence of grade D oocytesdecreased (P < 0.01) and the incidence of grade E oocytesincreased (P < 0.05) during the second phase compared tothe first one. No differences were found in terms of recoveryrate between the two phases.

The cleavage rate was significantly (P < 0.05) higher inthe second vs. the first phase (66.3% vs. 57.7%, respec-tively). Blastocyst rate was also higher (P < 0.01) in thesecond phase, both if it is calculated in relation to thetotal recovered COCs and, interestingly, on grade A + BCOCs. Consequently, a higher (P < 0.05) number of blasto-cysts/session/buffalo was obtained in the second vs. thefirst phase (Table 1).

3.2. Differences between I and II period

During the first 6 months a higher (P < 0.01) numberof total and small follicles/animal/PS was recorded com-pared to the last 3 months (Table 2). However, in the

last three months a higher (P < 0.01) incidence of mediumfollicles/animal/PS and a lower incidence of small folli-cles/animal/PS were recorded.

A higher (P < 0.01) number of all categories of COCs persubject/session was recorded during the first 6 months

G. Neglia et al. / Animal Reproduction Science 123 (2011) 180–186 183

Table 1Comparison between the first (63 days – 18 sessions; OPU carried out twice weekly) and the second (109 days – 16 sessions; OPU carried out once a week)phase in terms of COCs, follicles, in vitro embryo production and recovery rate (mean values/animal/session ± standard error).

Parameters I phase – 18 sessions (mean ± SE (%)) II phase – 16 sessions (mean ± SE (%))

Total follicles 5.65 ± 0.22A 4.45 ± 0.18B

Large follicles (size >1 cm) 0.50 ± 0.05A 0.69 ± 0.06B

(%) (8.8)A (15.5)B

Medium follicles (1 cm < size < 0.5) 1.36 ± 0.12A 0.81 ± 0.08B

(%) (24.1) (18.2)Small follicles (size < 0.5 cm) 3.79 ± 0.22A 2.95 ± 0.18B

(%) (67.1) (66.3)COCs 2.93 ± 0.20a 2.22 ± 0.19b

Grade A COCs 0.74 ± 0.07 0.66 ± 0.08(%) (25.2) (29.7)Grade B COCs 0.91 ± 0.10 0.72 ± 0.09(%) (31.1) (32.4)Grade C COCs 0.77 ± 0.09 0.66 ± 0.09(%) (26.3) (29.7)Grade D COCs 0.50 ± 0.07A 0.11 ± 0.03B

(%) (17.1)A (5.0)B

Grade E COCs 0.01 ± 0.01a 0.07 ± 0.02b

(%) (0.3)a (3.2)b

COCs A + B 1.65 ± 0.14 1.38 ± 0.13(%) (56.3) (62.1)Blastocyst 0.25 ± 0.06a 0.47 ± 0.08b

A B

< 0.05; A

Blastocyst/total COCs (%) 8.3Blastocyst/grade A + B COCs (%) 13.0A

Recovery rate (%) 53.80

Values with different letters, within rows, are significantly different (a,bP

compared to the last 3 months (Fig. 1). However, the inci-dence of different categories of COCs/animal/PS was notdifferent between the periods, with the exception of grade

E COCs/animal/PS, which was lower in the first period com-pared to the second one (Table 2).

Blastocyst production of the first period was 0.36 blas-tocyst/buffalo/session while no embryos were producedduring the 12 session of the second period. No blastocyst

Table 2Comparison between the first (6 months/34 sessions) and the second (3 monthproduction and recovery rate (mean values/animal/session ± standard error).

Parameters I period – 34 sessions (m

Total follicles 5.06 ± 0.15A

Large follicles (size >1 cm) 0.59 ± 0.04(%) (11.7)Medium follicles (1 cm < size < 0.5) 1.09 ± 0.08(%) (21.5)a

Small follicles (size < 0.5 cm) 3.38 ± 0.14A

(%) (66.8)A

COCs 2.58 ± 0.14A

Grade A COCs 0.70 ± 0.05a

(%) (27.1)Grade B COCs 0.81 ± 0.07A

(%) (31.5)Grade C COCs 0.72 ± 0.06A

(%) (27.9)Grade D COCs 0.31 ± 0.04A

(%) (12.0)Grade E COCs 0.04 ± 0.01A

(%) (1.5)A

COCs A + B 1.51 ± 0.10A

(%) (58.5)Blastocyst 0.36 ± 0.05A

Blastocyst/total COCs (%) 14.6Blastocyst/grade A + B COCs (%) 22.2Recovery rate (%) 49.21A

Values with different letters, within rows, are significantly different (a,bP < 0.05; A

21.432.1B

49.11

,BP < 0.01).

rate was generated during the second period, even if thepercentage of grade A + B COCs was similar to that recordedduring the first period (Table 2). In the last period cleav-

age rate was also poor (25.7% vs. 61.2%, in the second andthe first period, respectively). It is worth pointing out thatduring this unproductive period the efficiency of the lab-oratory on the slaughterhouse oocytes remained normal(>25% blastocysts/COCs A + B).

s/12 sessions) period of OPU in terms of COCs, follicles, in vitro embryo

ean ± SE (%)) II period – 13 sessions (mean ± SE (%))

3.71 ± 0.20B

0.59 ± 0.08(15.9)1.05 ± 0.11(28.3)b

2.07 ± 0.17B

(55.8)B

1.56 ± 0.15B

0.50 ± 0.06b

(32.0)0.44 ± 0.08B

(28.2)0.38 ± 0.07B

(24.4)0.09 ± 0.03B

(5.8)0.15 ± 0.04B

(9.6)B

0.94 ± 0.12B

(60.2)0.00B

0.000.0040.16B

,BP < 0.01).

184 G. Neglia et al. / Animal Reproduction Science 123 (2011) 180–186

COCs) an

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Fig. 1. Number of total follicles (FL), cumulus oocyte complexes (

. Discussion

The OPU technology has been largely used in order tonhance genetic improvement in farm animals (Galli et al.,001). This trial represents the longest experience of OPUver carried out in buffalo species. After the first period63 days), during which OPU was carried out twice weekly,t was necessary to increase the interval between oocyteampling from 3–4 to 6–7 days, because a reduction inollicle number occurred from the 13th PS. Nevertheless,uring the second phase (OPU performed once/week; 16S) a further reduction of follicles and COCs was observedompared to the first phase (OPU performed twice/week;8 PS). These results are in agreement with a previousxperience carried out in buffaloes in the same period ofhe year (Di Palo et al., 2001), when a decrease in theumber of follicles occurred after 60 days of OPU treat-ent. On the contrary, Boni et al. (1996), did not show

ny difference in the number of follicles and COCs in deepnoestrus buffaloes at more than 500 days open, over 4onths OPU carried out from October to February. Since

he buffalo donors in our trial were in the same conditionsold non-lactating subjects at the end of their productivend reproductive career) it could be speculated that the dif-erent patterns observed in the follicular and, hence, oocyteopulation may be due to the different seasons in whichhe two studies were performed. In our trial OPU began in

arch and after only two months treatment, the reductionf the follicle number led to the necessity to perform thePU technique once a week. This would represent a furtheronfirmation of buffalo sensitivity to photoperiod (Zicarelli,997a; Campanile et al., 2009, 2010).

Following the increase in the intersession interval, theotal number of follicles/animal/PS in the second phase was

educed compared to the first one and resulted in a reducedumber of recovered COCs/animal/PS. The increase of the

ntersession interval caused an enhanced percentage ofarge follicles/animal/PS and this may account also for thereater incidence of Grade E oocytes/animal/PS. However,

d good quality COCs (A + B) throughout the experimental period.

it is more difficult to explain the reduced incidence of gradeD oocytes/animal/PS, which occurred despite the increasein the intersession interval. It may be hypothesized thatafter 2 months a slowing down of the follicular turnoveroccurs, as indicated by the reduced number of follicles, aswell as the decreased incidence of grade D oocytes. Thedelayed follicular growth may result in a later dominanceof large follicles, which would require more time to induceatresia of the subordinates and hence degeneration of theoocytes.

Furthermore, the higher cleavage and blastocyst pro-duction recorded in the second phase (OPU performed onceweekly) vs. the first phase (OPU performed twice weekly),would suggest that the delayed follicular growth resultedin an improved oocyte competence. This statement is fur-ther supported by the different blastocyst rates calculatedon grade A + B COCs that supposedly should have given sim-ilar blastocyst yield. On the other hand, it is not possible torule out that the improved oocyte competence recordedin the second phase, when the incidence of large follicleswas higher, is related to the presence of small follicles inearly atretic phase, that have been demonstrated to provideoocytes with high competence to develop into embryos invitro (Salomone et al., 1999; Smith et al., 1996).

However, as we could not divide the animals in twogroups according to the intersession interval, because ofthe high individual variability, we can only speculate thatwhen the follicular turnover slows down, as a result of OPUsampling, delaying the PS leads to improved oocyte com-petence. It is interesting to note that in previous studiesdifferent intersession intervals were compared and it wasobserved that extending the PS interval from 4 to 5 daysdetermines worsening of oocyte quality because of greaterincidence of atresia (Boni, 1997). Our finding that extending

the PS interval up to 7 days does not affect oocyte qualityand improves the competence is a further confirmation thatrepeated punctures result in slower follicular growth.

The main finding of this study is that OPU can be suc-cessfully carried out on non-lactating buffalo cows for

duction

G. Neglia et al. / Animal Repro

six months. Afterwards, although good quality COCs arestill recovered, cleavage rate is markedly reduced and noblastocysts are produced. This result indicates that oocytedevelopmental competence is seriously affected by pro-longing OPU over 6 months. In fact, the reduction offollicles, total COCs and Grade A and B COCs observed inthe last three months, could only account for a decreasednumber of blastocyst produced. In this case, despite a sim-ilar percentage of grade A + B COCs, no blastocysts wereobtained in the second period. We can rule out that thiswas due to problems inherent to the laboratory, becausewe used both abattoir-derived and OPU-derived oocytescollected from different donors to test the efficiency ofthe laboratory that in these cases was high (approxi-mately 25–30% blastocyst rate). This unexpected findingis not easily explainable. It was previously demonstratedin buffaloes that the increase of days open negativelyaffects follicular and oocyte population (Boni, 1997), aswell as the response to superovulation treatment in buf-falo species (Zicarelli, 1997b). In our trial the high numberof days open (>18 months) and the old age (older than14 years) of the animals, together with the repeated OPUtreatment, could have been responsible for the reducedfollicle number and for the loss in oocyte developmen-tal competence, although the morphological quality wasnot affected. It has been demonstrated in cattle that, whilethe follicle number decrease with advancing age, no dif-ferences in oocyte quality as assessed by morphologicalcriteria are seen as far as up to 17 years of age; however,no data on their developmental competence are reported(Katska and Smorag, 1984). Furthermore, a decline in fertil-ity has been observed before total depletion of the ovarianoocyte reservoir in most mammalian species (Armstrong,2001). Factors that contribute to this decline in fertil-ity include hypothalamic-pituitary abnormalities, ovarianendocrine deficiencies, impaired oviductal function con-tributing to fertilization failure, decreased endometrialreceptivity (capability of allowing embryo attachment) andoocyte abnormalities (Adams, 1975; Werner et al., 1991).Despite abundant evidence that oocyte defects are majorlimiting factors in developmental competence (Armstrong,2001), comparatively little is known about the specificnature of oocyte deficiencies responsible for declining fer-tility with advanced maternal age. We may speculate thatrepeated OPU-related follicular aspiration determines acontinuous anticipation of follicular waves emergency,resulting in a premature reproductive “aging” of the ani-mals, that were already fourteen years old at the beginningof the trial. Furthermore, it has been previously demon-strated in dairy cow that prolonging OPU treatment afterthe third month causes a reduction in the number ofrecruited follicles between the third and the sixth month(Boni et al., 1997a). Since the follicular population in buf-falo is around 20% of that recorded in bovine (Van Tyet al., 1989) and OPU was carried out for 9 months in ourtrial, it may be possible that an earlier follicular depletion

occurred. Unfortunately, the donors were slaughtered atthe end of the trial and for this reason it was not pos-sible to repeat the OPU treatment after a resting period.As we could not collect and examine the ovaries, wecannot rule out that the repeated OPU treatment caused

Science 123 (2011) 180–186 185

a chronic inflammation of the ovary that, due to theincreased number of collagen fibres in the tunica albug-inea (Kruip et al., 1994) or to the effect of prostaglandins,nitric oxide and other inflammation by-products, may rep-resent another possible cause of the embryo productionfailure. Although it is difficult to compare the superovula-tory treatment with a repeated OPU, it is worth noting thatin a recent trial performed on mice, it was demonstratedthat repeated ovulation decreased the amounts of mito-chondrial DNA and increased 8-hydroxydeoxyguanosinein oocytes (Miyamoto et al., 2010). This phenomenonnegatively affected fertilization and development fromtwo cells to blastocyst (Miyamoto et al., 2010). Furtherstudies are needed in order to comprehend this phe-nomenon.

Interestingly, the loss of oocyte developmental com-petence occurred when the daily light hours decreased(autumn), which coincides with the most favorable periodfor the reproductive activity of the species at our lati-tudes (Zicarelli, 1997a). Therefore, the prolonged OPU hada detrimental effect on the reproductive function of thedonors, regardless of the photoperiod. On the contrary, inIndia (Manjunatha et al., 2009) a higher number of fol-licles, recovered COCs and blastocyst/animal/session wasobserved in buffaloes undergoing OPU during the peakcompared to the low breeding season, although blastocystrate was not different. However, in this study OPU wasperformed twice a week for only 8 weeks during each sea-son and primiparous buffaloes at 90–120 days open wereutilized.

In conclusion, OPU can be successfully performed undera continuous regime for up to 6 mo in non-lactating buffalocows at the end of their productive and reproductive careerwith a satisfactory embryo production efficiency. However,after this period, although a certain percentage of goodquality COCs is still recovered, a loss of oocyte develop-mental competence is observed. Further studies are neededto comprehend if this finding is repeatable and reversibleafter a resting period. Moreover, there is evidence thatwhen the follicular growth slows down, as a result ofrepeated OPU puncturing, extending the intersession inter-val to once per week improves oocyte competence andin vitro embryo production.

Acknowledgements

This research was supported by FISR – Fondo Integra-tivo speciale ricerca – DM 17/12/02. We are grateful toSchering-Plough Animal Health and Intervet-Italia for tech-nical and financial support.

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