A.K. Goel

Effect of egg yolk levels and equilibration periods on freezability of Jamunapari buck semen

Semen ejaculates from adult Jamunapari bucks (2–4 year-old) maintained at the Central Institute for Research on Goats, Makhdoom (UP) were utilized for a study to find out the freezability of buck semen at different levels of egg yolk during different hours of equilibration period by conventional freezing protocol. The ejaculates were extended to maintain sperm concentration approximately 100 million per dose (0.25 ml) in Tris-Citric Acid- Fructose (TCF) diluent having 0, 5, 10, 15 and 20% (v/v) egg yolk and 6% (v/v) glycerol as a cryoprotectant. Filling and sealing of straws were done at 5oC in cold handling cabinet after 2, 3, 4 and 5 h of equilibration period followed by vapour freezing of straws for 10 min at 2 cm above the liquid nitrogen and finally plunged into liquid nitrogen. Post-thaw motility, live sperm count, sperm abnormalities, acrosomal integrity and hypo-osmotic swelling test were conducted to assess freezability. The post-thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo-osmotic swelling (HOS) positive spermatozoa differed significantly (P<0.05) at different levels of egg yolk and equilibration periods. Our results indicated that 10% egg yolk and 4 h of equilibration period is the best combination for semen freezing. The overall results showed that the inclusion of egg yolk significantly improved sperm post-thaw motility, indicating its beneficial effects during the freezing steps of cryopreservation. Furthermore, neither glycerol nor egg yolk alone protected the acrosome and tail membrane; the combination of two significantly (P<0.05) reduced the proportion of acrosome-damaged spermatozoa.

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non-reproductive tissues.

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high-fertile (G1) and low-fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real-time PCR (Roche LC-480). Our work shows that the relative quantification by RT-PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT-PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.

Zona free hamster ova penetration test: fertility indicator of buck spermatozoa

An experiment was conducted to assess the sperm fertility of Sirohi bucks. Ejaculates from three bucks were collected through artificial vagina and evaluated microscopically at 37° C. After final washing with Biggers-Whitten Whittingham (BWW) medium, semen was kept in a CO2 incubator for 20 min (swim up). The hamster oocytes were recovered following superovulation and visualized on the basis of cumulus cell mass. The oocytes were washed in BWW medium and then denuded with 0.1% hyaluronidase, the cumulus-free oocytes were treated with 0.1% protease for 2–3 min for zona lysis. Oocytes and swim up motile spermatozoa were co-incubated at 37° C for 3 h. in already incubated BWW drop (50 μl). At the end of co-incubation, washing of oocyte sperm complexes was done with BWW medium and then kept in hypotonic KCl solution (50 mg/ml), fixed in 2.5% glutaraldehyde for 10 min and stained with 0.1μg/ml DAPI (4,6-di amidino 2-phenyl indole). The number of sperms penetrated into the oocytes was determined. The result of the study indicated that among the three bucks, buck No. 1 had higher penetration rate (100%) as compared to buck No. 2 (93.00%) and buck No 3 (86.66%).

Development of parthenote following in vivo transfer of embryos in Capra hircus

The aim of this study is to generate parthenogenetic embryos from chemically activated in vitro matured caprine oocytes and to study the in vivo developmental potency of such embryos. The parthenogenetic embryos (2–8 and 16 cells to morula stage) were surgically transferred in 26 recipients. Pregnancy in recipients following embryo transfer was monitored by ultrasonography. The recipient aborted a foetus on day 34 post transfer. Sexing of parthenogenetic foetus showed a single band of amelogenin gene indicating female cell DNA. Microsatellite analysis revealed that the recipient has not contributed genetically to the parthenogenetic foetus confirming the identity of aborted foetus of parthenogenetic origin. The authors believe that this is the first authentic report on in vivo development of parthenogenetic foetus in Capra hircus.