Reproduction in Domestic Animals

Reproduction in Domestic Buffalo 203Reproductive TechnologiesArtificial inseminationThe procedures for processing and using buffalo semenfor AI are based mainly on techniques developed forcattle with some modifications (Vale 1997; IAEA 2005).The main differences are in the semen diluents, with Trisbuffers, egg yolk, skim milk and coconut water beingcommonly used as ingredients for preserving semen inboth chilled (+4°C) and deep-frozen forms. The cryoprotectiveagent used for freezing buffalo semen isglycerol, at a final concentration of 6.5–7.0%. Variousadditives have been investigated for possible beneficialeffects on buffalo spermatozoa during freezing andthawing, and the two proteins that help to maintainhigher post-thaw motility are oviductal proteins (Kumaresanet al. 2005) and Bradykinin (Shukla and Misra2007). Buffalo spermatozoa, subjected to freezing andthawing, appear to have a shorter fertile lifespan insidethe female tract than those in fresh semen (Moioli et al.1998). Thus, proper detection of heat and timing of AIbecome critical when frozen semen is used and may beone reason for the lower conception rates, while anothercould be the narrow cervix of the buffalo, which makesAI more difficult.Apart from the technical aspects, there are manyconstraints which influence the success of AI in buffalo,especially in developing countries. These include factorsthat impede the effective and timely delivery of services,such as poor communication, lack of transport andinadequate remuneration for AI technicians, and limitationsof smallholder farming systems, such as poornutrition and reproductive management of cows.Separation of X and Y spermatozoaIdentification of X- and Y-bearing spermatozoa inbuffalo can be performed by fluorescence in situhybridization, using the cattle Y-chromosome-specificBC1.2 probe or the X- and Y-specific probes from theyak (Re´vay et al. 2003). Use of high-speed flowcytometric cell sorting, followed by deep AI carriedout near the utero-tubal junction (UTJ) with 2.5 millionlive frozen-thawed sperm, resulted in 43% conceptionrate with eight out of nine fetuses corresponding to thepredicted sex (Presicce et al. 2005). Semen depositionnear the UTJ of buffalo can be performed using theGhent device that was developed for cattle (Presicceet al. 2004). Sex-sorted sperm has also been usedsuccessfully for in vitro fertilization (IVF) and embryotransfer (Lu et al. 2007).Oestrous synchronizationThe procedures used in the past for oestrous synchronizationin buffalo were empirically based on thosedeveloped for cattle, aimed at either inducing prematureluteolysis using prostaglandins or prolonging the lutealphase using progestagens (Perera 1987). However, theefficacy of prostaglandins for synchronizing ovulation isnow known to be dependent upon progesterone concentrationand ovarian follicular status at the time oftreatment (Brito et al. 2002). Use of the two-dose regimeof prostaglandin overcomes some of the above limitations,but manipulation of follicular development isnecessary to achieve better synchrony and improvedfertility (De Rensis and Lo´pez-Gatius 2007). Therefore,most current protocols include GnRH or gonadotrophinsin combination with prostaglandins, progesteroneor oestradiol. The ‘Ovsynch’ protocol (GnRH, prostaglandin7 days later and second GnRH 2 days later) hasbeen successful in synchronizing ovulation in 70–90% ofbuffalo with conception rates ranging from 33% to 60%(Baruselli et al. 1999; Paul and Prakash 2005). Inaddition to the type of protocol selected, the followingfactors must also be addressed to achieve success inbuffalo: (a) selection of animals that are in goodnutritional condition and free from disease; (b) preventionof stress during the procedures for treatment andAI, especially under tropical conditions, where animalsmay be herded together or moved to other locations;and (c) where seasonal differences exist, schedulingtreatment for the more favourable periods when themajority of animals are cycling.Multiple ovulation embryo transferThe current status of MOET and other advancedreproductive technologies in buffalo has been recentlyreviewed (Drost 2007). Studies in many countries haveconfirmed that buffalo have a lower superovulatoryresponse than cattle, attributed mainly to the smallerpopulation of recruitable follicles in the ovary (Madanet al. 1996; Manik et al. 2002). A further limitationappears to be the relatively low rate of transfer ofoocytes to the oviduct and ⁄ or impaired transport of ovaand embryos in the reproductive tract (Baruselli et al.2000). In a large scale MOET operation in India (Misraet al. 1994), the total embryo yield per treatmentincreased from 1.77 to 3.83 over 5 years, and thenumber of viable embryos from 0.92 to 2.13; thetransfer of 469 embryos resulted in a pregnancy rateof 17% and a calving rate of 9.8%. In Italy, Campanileet al. (1995) transferred 76 buffalo embryos andobtained a pregnancy rate of 30%, with no significantdifference between fresh or frozen-thawed embryos andbetween morulae or blastocysts. Yet, the overall rate ofsuccess in terms of calves born per superovulation is stilltoo low for MOET to be widely applicable under fieldfarmingconditions. Two situations in which it mighthave a role are for establishing nucleus breeding stocksor for conservation of genetic resources.In vitro embryo production and cryopreservationThe low efficiency of MOET in buffalo has led to anincreased interest in in vitro embryo production (IVEP)technologies for achieving rapid genetic improvement.The sequence of procedures involving recovery ofoocytes from ovaries of slaughtered animals by directaspiration or from live animals by ultrasound-guidedtransvaginal ovum pick-up (OPU), followed by in vitromaturation, IVF and in vitro culture have been studiedin buffalo over the past decade (Boni et al. 1996; Madanet al. 1996; Hufana-Duran et al. 2004), but the successÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag