New Control of Buffalo Follicular Dynamics for Artificial … · 2013. 11. 20. · Ovarian...

Control of Buffalo Follicular Dynamics for Artificial Insemination, Superovulation and In Vitro Embryo Production

Pietro Sampaio BARUSELLIa*, J.G. SOARESa, L.U. GIMENESb, B.M. MONTEIROa, M.J. OLAZARRIa

and N.A.T. CARVALHOc aUniversidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Reprodução Animal, São Paulo – SP, 05508-240, Brazil bDepartamento de Medicina Veterinária Preventiva e Reprodução Animal, FCAV- UNESP, Jaboticabal – SP, Brazil cUnidade de Pesquisa e Desenvolvimento de Registro - Pólo Regional do D.S.A. do Vale do Ribeira/APTA, Registro-SP, Brazil

*Corresponding email: [email protected] ABSTRACT

Currently, timed ovulation induction and timed artificial insemination (TAI) can be performed in buffalo using GnRH or estradiol plus progesterone/progestin (P4)-releasing devices and prostaglandin F2α (PGF2α). The control of the emergence of follicular waves and of ovulation at predetermined times, without the need for estrus detection, has facilitated the management and improved the efficiency of AI programs in buffalo during the breeding and nonbreeding season. Multiple ovulations, embryo transfer, ovum collection and in vitro embryo production have been shown to be feasible in buffalo, although low efficiency and limited commercial application of these techniques have been documented as well. These results could be associated with low ovarian follicular pools, high levels of follicular atresia and failures of the oocyte to enter the oviduct after superstimulation of follicular growth. This review discusses a number of key points related to the manipulation of ovarian follicular growth to improve pregnancy rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in buffalo. Keywords: artificial insemination, buffalo reproduction, embryo production INTRODUCTION

The understanding of follicular dynamics in buffalo is necessary for developing new techniques and improving the currently used regimens for the manipulation of the estrous cycle. Ovarian follicular dynamics in buffalo are similar to those of cattle. Although the 2-wave cycle is the most common in buffalo (63.3%), both 3-wave and 1-wave cycles have also been observed. The number of waves in a cycle is also associated with the luteal phase and with the estrous cycle length. However, the number of follicles recruited per follicular wave is lower in buffalo than in cattle (Baruselli et al., 1997; Gimenes et al., 2009, Campanile et al., 2010). The success of several reproductive programs is closely related to ovarian follicular development and ovulation. In recent decades, several therapies have been proposed for manipulating ovarian follicle growth in buffalo. These hormonal manipulations have been successfully used to optimize the reproductive outcomes following the application of various biotechnologies.

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Buffalo Bulletin 2013 Vol.32 (Special Issue 1): 160-176

Accepted April 10, 2013; Online November 11, 2013.

Timed artificial insemination (TAI) programs provide an organized approach to the enhanced use of artificial insemination (AI), the progress of genetic gain and the improved reproductive efficiency of cattle in dairy and beef herds (Pursley et al., 1995; Baruselli et al., 2004). The success of biotechnologies such as TAI depends on the evolution of ovarian follicular manipulation techniques. In buffaloes, hormonal treatments have been designed to control both luteal and follicular functions, providing exciting possibilities for the synchronization of follicular growth and ovulation that can enable the use of timed artificial insemination (TAI) during the breeding and nonbreeding season (Singh et al., 1988; Baruselli et al., 1999a; Neglia et al., 2003a; De Rensis et al., 2005). Satisfactory pregnancy rates of approximately 40% to 60% (Baruselli et al., 1999b; Berber et al., 2002; Neglia et al., 2003a; Paul and Prakash, 2005; Ali and Fahmy, 2007) have been achieved with the Ovsynch protocol (Day 0, GnRH; Day 7, PGF2α; Day 9, GnRH; TAI 16 hours after the second GnRH injection; Pursley et al., 1995) in cycling buffalo synchronized during the breeding season. However, anestrous buffalo respond poorly to the Ovsynch protocol and have lower pregnancy rates after TAI during the nonbreeding season (Baruselli et al., 2003a; De Rensis et al., 2005; Ali and Fahmy, 2007; Baruselli et al., 2007). However, treatment with intravaginal P4 devices followed by eCG at device removal has been used to increase ovulation rates, CL growth rate, initial P4 concentrations, and pregnancy rates after TAI in buffalo during the nonbreeding season (Carvalho et al., 2013).

The use of superovulation (SOV) followed by AI is a technique that generates greater numbers of embryos per donor in cattle (Mapletoft et al., 2002). These techniques, which are associated with embryo transfer (ET) to recipients, are powerful tools that facilitate the dissemination of genetic material of high quality (Bó et al., 2002; Baruselli et al., 2011). However, buffalo donors generally have lower embryo recovery rates than bovines. While buffaloes have shown follicular responses after superovulation treatment (mean of 15 follicles > 8 mm), only a moderate ovulation rate (approximately 60%) and CL yield at the time of flushing (approximately 9 CL) and low embryo recovery rates (34.8%; Baruselli et al., 2000) have been obtained. The embryo recovery rate in buffaloes (approximately 20 to 40%) is lower than in bovines (63 to 80%; Boland et al., 1991; Adams, 1994; Vos et al., 1994; Shaw et al., 1995). This divergence in embryo recovery rates was hypothesized to be related to a failure of oocyte capture and/or of oocyte transport along the oviduct (Baruselli et al., 2000).

Ovum pick up (OPU), associated with in vitro embryo production (IVP), is another interesting technology that produces embryos from selected donors (Boni et al., 1996; Neglia et al., 2003b, Sá Filho et al., 2009). This technology has the potential to enhance genetic progression through the female lineage in buffaloes. The success of OPU–IVP is directly related to oocyte quantity and quality. The use of this technology in buffaloes has been limited by the innately low number of follicles and hence of cumulus–oocytes complexes (COCs) that can be recovered per ovary (Gasparrini, 2002; Gimenes et al., 2010), as well as by the seasonality of follicle production (Di Francesco et al., 2011).

This review discusses a number of key points relating to the manipulation of ovarian follicular growth to improve conception rates following TAI and ET of in

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vivo- and in vitro-derived embryos in buffalo. The discussion focuses on the control of buffalo follicular dynamics for artificial insemination, multiple ovulation, embryo transfer (MOET), ovum pick up and in vitro embryo production.

CONTROL OF BUFFALO FOLLICULAR DYNAMICS FOR ARTIFICIAL INSEMINATION

Artificial insemination (AI) is one of the major biotechnologies used in domestic species and an important tool for the dissemination of superior genetic material of paternal origin. However, the use of this technique in the conventional manner (i.e., estrus detection following AI) presents two significant difficulties in buffaloes. The first is related to inefficient estrus detection due to discrete estrous behavior. The second is related to the seasonal and nutritional anestrous that lead to decreased reproductive activity in the species.

Hormonal protocols have been developed to control ovarian follicular dynamics and allow the use of AI without heat detection. In recent years, our research group developed studies to evaluate the efficiency of the Ovsynch protocol in buffalo (D0: GnRH; D7: PGF2α; D9: GnRH; TAI 16 hours after the 2nd GnRH injection; Baruselli et al., 1999a, 1999b; Berber et al., 2002; Baruselli et al., 2003a). In these experiments, we confirmed that buffaloes respond to hormonal treatment and that a new follicular wave emerges due to the ovulation of the dominant follicle present at the time of the first administration of GnRH. On day 7, buffaloes respond to PGF2α (luteolysis), and on day 9, approximately 80% of animals experience a synchronized ovulation within 12 hours. Additionally, a pregnancy rate (PR) of 50% can be obtained in cycling buffaloes during the breeding season. Nevertheless, PR is influenced by body condition score (BCS; good efficiency is achieved when BCS ≥ 3.5; 1 to 5 scale), parity (primiparous animals have lower PR than multiparous), and period of the year (higher PR is observed during the breeding season than in the non-breeding season).

Carvalho et al. (2007a) documented an increase in the PR and birth rates with the administration of GnRH 6 days after TAI in buffaloes on the Ovsynch protocol. This GnRH administration induced the formation of an accessory CL in buffaloes. The accessory CL increased the plasmatic concentration of progesterone (P4) and resulted in a positive effect on the PR and birth rates (Campanile et al., 2010; Marques et al., 2012). Studies have shown that P4 controls the function and secretion of the uterine glandular system (Spencer et al., 2004), which is important for the nutrition and development of the embryo (Mann et al., 1999). Therefore, the production of accessory CLs in buffalo represents an alternative method of increasing the efficiency of TAI.

When the Ovsynch protocol is used during the nonbreeding season (spring and summer; period of high anestrous incidence), lower PRs are obtained (approximately 7 to 30%; Baruselli et al., 1999b and Baruselli et al., 2002a). Therefore, studies have been conducted to develop the use of different hormonal protocols to increase PR in buffaloes submitted to TAI during seasonal anestrous. Various progesterone-releasing intravaginal devices impregnated with different amounts of progesterone (0.5 to 1.9 g) are commercially available. These devices contain natural progesterone, and blood levels of 4 to 5 ng/ml of progesterone are

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reached during their use. Lower doses (3 to 6 mg) can be used with auricular implants of norgestomet, which is more potent than natural progesterone.

Progesterone protocol involves the insertion of an intravaginal progesterone device or a norgestomet implant and intramuscular injections of estradiol benzoate (EB) on a random day of the estrous cycle (day 0). Nine days later (day 9), the device/implant is removed and intramuscular doses of PGF2α and eCG are administered. Forty-eight hours later (day 11), ovulation is induced by the administration of GnRH or hCG. TAI is performed 16 hours after the induction of ovulation (Baruselli et al., 2003b; Carvalho et al., 2007b).

The combination of progesterone/progestin and estradiol at the beginning of the protocol (day 0) is effective in inducing the emergence of a new follicular wave due to the suppression of both FSH and LH which promote the atresia of all follicles present in the ovary in cattle (reviewed by Bó et al., 2003) and in buffaloes (reviewed in Baruselli et al., 2007). Previous studies carried out in postpartum anestrous cows have demonstrated that P4 treatment stimulates an increase in LH pulse frequency during and following the treatment period (Rhodes et al., 2002). Treatment of anestrous cows with P4 results in greater follicular fluid volume and circulating concentrations of estradiol, increased pulsatile release of LH and increased numbers of LH receptors in granulosa and theca cells in preovulatory follicles (Rhodes et al., 2002). Furthermore, a short period of elevated P4 concentrations during the anestrous period is important for the expression of estrus and for subsequent normal luteal function (McDougall et al., 1992).

The use of equine chorionic gonadotropin (eCG) at the time of the removal of the progesterone-releasing device resulted in increased ovulation and pregnancy rates in suckled cows treated during postpartum anestrus (Baruselli et al., 2004). In buffaloes, treatment with eCG at the time of device removal increases the diameter of the dominant follicle at TAI (13.7±0.4 vs. 12.6±0.6mm, P=0.09) and the ovulation rate (66.7 vs. 44.8%, P=0.05); at the subsequent diestrus, treatment results in increased CL diameter (15.8±0.92 vs. 12.7±0.77 mm, P=0.03), increased P4 concentrations (0.59±0.08 vs. 0.27±0.05 ng/mL, P=0.01) and increased PRs (52.7 vs. 39.4%, P=0.03; Carvalho et al., 2013). These results confirm the necessity of eCG in ovulation synchronization protocols for TAI during the non-breeding season. In summary, eCG treatment stimulates the growth and maturation of the dominant follicle, resulting in increased ovulation rates and progesterone production by the subsequent CL and greater PR after TAI in anestrous buffalo.

Carvalho et al. (2012b) evaluated the possibility of replacing the ovulation inducer (GnRH) with EB in the buffalo TAI protocol. Treatment with BE resulted in a satisfactory follicular response, ovulation rate and PR in buffaloes synchronized for TAI during the nonbreeding season.

The use of the Ovsynch protocol during the breeding season and of P4+EB, PGF2α and eCG during the nonbreeding season resulted in a PR of approximately 50% in a single TAI. Therefore, the TAI program can be used throughout the year to efficiently schedule conception and the calving period in buffalo.

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CONTROL OF BUFFALO FOLLICULAR DYNAMICS FOR MULTIPLE OVULATION AND EMBRYO TRANSFER (MOET)

Despite the progress achieved with TAI and the successful application of this technique in buffalo herds, the biotechnologies used to increase the dissemination of maternal genetic material still have low diffusion. This can be attributed to unsatisfactory numbers of embryos obtained per buffalo donor and reflect the only partial understanding of the events involved in the manipulation of the estrous cycle, follicular superstimulation and ovulation in this species.

Although births of buffaloes achieved using MOET have been reported in several countries (Drost et al., 1983; Drost et al., 1988; Misra et al., 1990; Zicarelli, 1992; Baruselli, 1994), the use of this technique still has limitations, mainly related to the low embryo recovery rate per donor (Misra et al., 1990; Ambrose et al., 1991; Baruselli, 1994; Zicarelli et al., 1994; Taneja et al., 1995; Madan et al., 1996; Zicarelli et al., 2000; Baruselli et al., 2000; Carvalho et al., 2002). Currently, superovulated buffaloes produce on average 1-3 viable embryos for harvest. This average remains lower than the mean number of recovered embryos in the bovine (10 total and 6 transferable embryos; Boland et al., 1991).

The advances provided by the MOET technique have revealed that buffaloes have satisfactory responses to superovulatory treatment (Baruselli, 1997), although embryo recovery in buffaloes is less efficient than in cows (Baruselli et al., 2000). A low number of embryos recovered in buffaloes has also been described by several authors (Karainov, 1986; Madan, 1990; Drost, 1996; Zicarelli, 1997). According to Baruselli et al. (2000), only 34.8% of buffalo ovulations obtained through superstimulation of follicular growth resulted in recovered embryonic structures, a percentage much lower than that found in bovines by Adams (1994) who recorded rates of 63% to 80%. This disparity between embryo recovery rates may be related to failures in the collection and/or transportation of oocytes in the oviduct.

According to Hunter (1988), the mechanisms involved in oocyte transportation (ciliary beats of the epithelium of the oviduct and waves of contraction of the myosalpinx) are controlled by ovarian steroids.

The low number of embryos obtained in buffalo MOET could be attributed to high estrogen (E2) levels during the superovulation treatment, as postulated by Misra et al. (1998). Prolonged exposure to elevated concentrations of 17β-estradiol may change the intrauterine and/or oviductal environment and, consequently, impair normal embryonic development and transport. It is also possible that buffalo are more sensitive to high 17β-estradiol levels during superstimulation treatments than bovines (Beg et al., 1997). To test this hypothesis, our group conducted experiments to reduce estradiol levels during superstimulation treatments (Baruselli et al., 2002b; Carvalho et al., 2002) using exogenous sources of progesterone during preovulatory periods or deslorelin bioimplants during superovulation. However, no increase in embryo recovery rate was observed.

To further test this hypothesis, our group performed a sequence of trials to investigate in detail the anatomical and physiological inter-relationships between ovarian steroids and the genital system of buffaloes and bovines (Carvalho et al., 2011; Carvalho et al., 2012a). We studied morphometric characteristics of females with single or multiple ovulations and we compared the direction of ciliar

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movements in oviducts exposed or not exposed to estradiol in the culture medium. We observed no effect of estradiol on embryo transport or on ciliar movement of oviducts. However, we found that buffaloes have a higher number of anovulatory follicles, a more rigid ovary-mesovarium connection, and a thicker infundibulum muscle layer than bovine females. These factors could be partially responsible for the low embryo recovery rates in buffaloes.

As previously mentioned, low oocyte quality in buffaloes may be associated with a fragile connection between the oocyte and granulosa cells, in contrast to what occurs in bovine species (Gasparrini, 2002). Some studies infer that rbST can enhance this connection by a direct and/or indirect effect of IGF-1 and increase the population of small antral follicles (Pavlok et al., 1996; Lucy, 2000). Additionally, rbST can stimulate the expansion of cumulus cells (Izadyar et al., 1998), contributing to oocyte adhesion to fimbria and ciliated cells of the endosalpinx, which can improves embryo recovery in superstimulated animals. To confirm this hypothesis, studies were performed to investigate the effect of different doses of rbST (0, 250, or 500 mg) on embryo recovery in superovulated buffaloes (Baruselli et al., 2003c; Carvalho et al., 2007c). The results were mostly inconclusive; in the first study, 500 mg of rbST increased embryo recovery (50.0 vs. 33.3%; P=0.06) and the number of structures recovered (5.1±6.8 vs. 1.6±1.7; P=0.18); however, in the second experiment, none of the doses of rBST used had any impact on the efficiency of MOET in buffalo.

The low embryo recovery rate reported in buffaloes may be related to the failure of oocytes to enter the oviduct after superstimulation of follicular growth (Baruselli et al., 2000). In rabbits, the administration of sequential doses of PGF2α during the periovulatory period stimulated the contraction of oviduct smooth muscles, allowing the activation of the oviduct fimbriae to capture the oocytes (Osada et al., 1999). Based on this observation, our research group recently (Soares, et al., 2013) verified that the use of PGF2α during the periovulatory period increases the total number of structures (ova and embryo) recovered in superovulated buffaloes (buffalo treated with PGF2α = 3.5±0.6 vs. control group = 2.3±0.5; P=0.02). Furthermore, the treatment with PGF2α increased the number of transferable (1.8±0.5 vs. 2.7±0.6; P=0.05) and freezable embryos (1.8±0.5 vs. 2.6±0.6; P=0.08; Figure 1). The results indicate that the administration of PGF2α during the periovulatory period was effective at increasing embryo production (total ova/embryos recovered) in superovulated buffaloes.

Generally, the reason for the low embryo recovery rate in superovulated buffaloes remains unknown, compromising the efficiency and the application of embryo transfer technology in this species. Further studies are needed to enable the use of MOET in buffalo, to allow this technique to be widely used by farmers and to accelerate genetic gain and productivity of buffalo herds. CONTROL OF BUFFALO FOLLICULAR DYNAMIC FOR OVUM PICK UP AND IN VITRO EMBRYO PRODUCTION

Due the variable results of in vivo embryo recovery in superovulated buffaloes, the association of ovum pick up (OPU) with in vitro embryo production (IVP) represents an alternative method of exploiting maternal genetics. Historically,

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OPU-IVP in buffaloes produced lower outcomes (Gasparrini, 2002; Ferraz et al., 2005; Sá Filho et al., 2009; Gimenes et al., 2010) than in bovines (Lonergan & Fair, 2008, Pontes et al., 2011). However, recent studies have demonstrated the commercial potential of these techniques in the buffalo species.

Two main biological problems seem to be related to the inefficiency of the OPU-IVP technique in buffaloes: low numbers of follicles on the ovary, witch influence directly the numbers of oocyte recovery per OPU, and poor oocyte quality (only 27.3% to 31.3% of recovered oocytes are classified as viable; Campanile et al., 2003).

The first problem can be related to the lower number of follicles recruited per follicular wave (Baruselli et al., 1997), as observed in studies comparing buffaloes with Bos indicus cattle (Ohashi et al., 1998; Gimenes et al., 2010). Additionally, a higher level of follicular atresia was reported (Danell, 1987; Le Van Ty et al., 1989) and, consequently, a lower number of total recoverable and viable oocytes. Buffaloes and cattle raised with contemporary nutrition and management were compared post mortem by Ohashi et al. (1998), and in vivo by Gimenes et al. (2010; Table 1). In both studies, lower number of follicles (4.5±1.9 vs. 8.7±5.9; Ohashi et al., 1998) and viable oocytes (2.2±1.1 vs. 4.1±2.6; Ohashi et al., 1998) were observed in buffaloes than in Bos indicus cattle.

The second problem with the OPU-IVP technique in buffaloes can be attributed to a more fragile zona pellucida (Mondadori et al., 2010) and a more fragile bonding between cumulus cells and the oocyte (Ohashi et al., 1998; Gasparrini, 2002) in buffaloes than in cattle.

To improve oocyte quality and recovery, the following studies were conducted by our group. Initially, we tested the hypothesis that bST could elevate circulating IGF-1 levels, promoting recruitment of a greater number of follicles and enhancing oocyte quality (Sá Filho et al., 2009). OPU was performed twice a week for 10 sessions in control and bST (500 mg) groups. The bST treatment resulted in greater numbers of aspirated follicles (12.2 ± 0.1 vs. 8.7 ± 0.04) and retrieved oocytes per donor per session (5.2 ± 0.5 vs. 4.1 ± 0.5) than the control treatment. However, when oocytes were submitted to IVP procedures, a similar blastocyst formation rate was obtained for animals in the control (26.0%) and bST group (19.7%). In this study, 57 embryos were produced in vitro and transferred fresh (n = 7) or vitrified (n = 25) into 32 previously synchronized recipients (GnRH/PGF2α/GnRH; Sá Filho et al., 2005). Pregnancy rates obtained were 14.3% (1/7) for fresh embryos and 8.0% (2/25) for vitrified embryos. The association of OPU and IVP resulted in the first births from fresh (n=1) and vitrified (n=2) embryos reported in North or South America.

In a subsequent trial (Ferraz et al., 2007), the effects of the intervals between OPU (once a week or every 14 days) and the use of bST (control or treated with 500 mg of rbST) were evaluated. The treatment with bST increased (P<0.05) the number of visualized follicles (15.3 ± 0.5 vs. 12.1 ± 0.4), but reduced (P<0.05) the number of blastocysts (0.8 ± 0.1 vs. 1.4 ± 0.2) and the blastocyst formation rate on day 7 (10.9% vs. 18.9%). On average, OPU performed once a week reduced (P<0.05) the number of aspirated follicles (12.8 ± 0.4 vs. 15.6 ± 0.7) and the number of total (8.5 ± 0.3 vs. 10.0 ± 0.5) and viable oocytes (6.1 ± 0.3 vs. 7.2 ± 0.4).

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To test an alternative approach, the effect of synchronization of follicular wave emergence on OPU-IVP in buffalo, Nelore (Bos indicus) and Holstein (Bos taurus) heifers raised under conditions of contemporary management and nutrition was evaluated (Gimenes et al., 2010). An important factor that is known to directly influence the quantity and quality of oocytes obtained by OPU and, consequently, IVP, is the phase of the estrous cycle (Machatková et al., 1996; Hendriksen et al., 2000; Machatková et al., 2000; Merton et al., 2003; Vassena et al., 2003). Oocytes from follicles with a mild degree of atresia have higher rates of embryonic development and require less time for in vitro maturation (Wit et al., 2000; Hendriksen et al., 2000; Vassena et al., 2003). In our study, animals were synchronized to be aspirated 1, 3 or 5 days after the follicular wave emergence. The results of this experiment show that OPU-IVP is less efficient in buffalo and Holstein heifers than in Nelore heifers (Table 1). However, contrarily to our initial hypothesis, the synchronization of follicular emergence for OPU did not affect IVP in any of the three genetic groups.

Recently, the influence of season (winter or summer) on oocyte viability (number of viable oocytes and mitochondrial DNA amount) was investigated in nulliparous (n = 8) and multiparous (n = 8) buffaloes (Macabelli et al., 2012). All animals had synchronized follicular wave emergences by hormonal treatment before OPU was performed five days after the beginning of the protocol (progesterone plus estradiol benzoate). Before OPU was performed, cutaneous and rectal temperatures and the respiratory frequency of all animals were measured. Oocytes (winter = 22 of nulliparous and 13 of multiparous; summer = 21 of nulliparous and 15 of multiparous) were processed for quantification of mitochondrial DNA (mtDNA). As expected, cutaneous and rectal temperatures and respiratory frequency were higher in summer than in winter (P> 0.05). Although the number of viable oocytes was greater in nulliparous animals than in multiparous animals (13.4±2.2 vs. 6.3±0.8), the percentage of viable oocytes was lower in summer than in winter (55.5±3.6 vs. 64.4±2.6). The number of copies of mtDNA is shown in Figure 1. Although mtDNA analyses do not suggest a negative effect of summer on oocyte viability, further conclusions on the effect of seasonality on oocyte competence are expected from ongoing gene expression analyses.

Finally, a partnership between a Brazilian private laboratory of IVP (Cenatte Embriões LTDA) and universities (UFMG, USP, and UNINA) proved that the production of buffalo embryos in vitro for commercial purposes was possible (Saliba et al., 2011; 2012; 2013a; 2013b). Briefly, buffalo donors were submitted to OPU sessions every 20 – 30 days from July to December, 2010. Viable oocytes (grade 1, 2 and 3) were subjected to IVP procedures, resulting in a blastocyst production rate of 44.9% (262/584). A portion of the embryos produced was transferred fresh or vitrified, resulting in pregnancy rates 30 days later of 43.5% (50/115) and 37.1% (26/70), respectively. Embryonic mortality 60 days after embryo transfer was 4% (2/50) and 5.7% (4/70), respectively. The results obtained in these studies were higher than those previously described in the literature and demonstrate the potential of OPU-IVP in buffaloes.

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174


Tab

le 1

. Ef

fect

of g

enet

ic g

roup

(Nel

ore

– Bo

s ind

icus

, Hol

stei

n –

Bos t

auru

s and

Buf

falo

– B

ubal

us b

ubal

is) o

n O

PU-I

VP

(Gim

enes

et a

l., 2

010)

.

N

elor

e (B

os in

dicu

s)

(n=9

)

Hol

stei

n (B

os ta

urus

) (n

=9)

Buf

falo

(B

ubal

us b

ubal

is)

(n=9

) M

ean

num

ber o

f vis

ualiz

ed fo

llicl

es

41.0

a ± 2

.1

22.1

b ± 1

.3

18.8

b ± 0

.9

Mea

n nu

mbe

r of t

otal

ooc

ytes

37

.1a ±

2.5

15

.4b ±

1.2

14

.8b ±

1.0

R

ecov

ery

rate

(%)

89.4

a 73

.3b

82.8

ab

Mea

n nu

mbe

r of v

iabl

e oo

cyte

s 30

.8a ±

2.4

10

.7b ±

1.0

7.

9b ± 0

.7

Mea

n n

umbe

r of s

truct

ures

in in

vitr

o cu

lture

18

.7a ±

0.8

8.

0b ± 0

.5

7.5b ±

0.4

M

ean

num

ber o

f cle

aved

stru

ctur

es

15.4

a ± 0

.7

4.6b ±

0.4

4.

4b ± 0

.3

Cle

avag

e ra

te (%

) 81

.8a

59.1

b 62

.3b

Mea

n nu

mbe

r of b

last

ocys

ts o

n D

7 5.

1a ± 0

.6

1.0b ±

0.2

0.

6b ± 0

.1

Bla

stoc

yst r

ate

(%)

25.8

a 13

.6b

9.1b

Mea

n nu

mbe

r of h

atch

ed b

last

ocys

ts

2.6a ±

0.4

0.

3b ± 0

.1

0.3b ±

0.1

H

atch

ing

rate

(%)

50.6

a 23

.2b

31.9

ab

a ≠

b: su

pers

crip

t let

ters

in th

e sa

me

row

diff

er (P

<0.0

5).

175


Figure 1. Total number of structures (ova and embryo recovered), transferable, and freezable embryos of buffalo donors (n=22) treated with sequential doses of PGF2α during the periovulatory period.

Figure 2. Number of copies of mitochondrial DNA (mtDNA) in oocytes of

nulliparous and multiparous animals during winter and summer. *The asterisk indicates a significant difference (P <0.05) between seasons within the same animal category.

176


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