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A cDNA microarray containing 43,661 differentially expressed genes was carried out on the blastoderm of fertilized and facultative parthenogenic turkey embryos at different hours of development. The total number of up-regulated (UR) and down-regulated (DR) genes at 0, 12, and 24 h of development were 725 and 1436, 942 and 599, and 589 and 1044, respectively. Common genes between 0 and 12 h, 12 and 24 h, and 0 and 24 h were 55, 67, and 110, respectively. The proportion of genes showing above 50-fold UR and DR at 0, 12, and 24 h of development were 2.0% and 1.5%, 0.5% and 1.2%, and 0.2% and 1.1%, respectively. Eight UR genes were validated (APOA1, THRAP3, ARL14EP, PSAP, MOG, MYBPC2, MTIF3 and EDG4) and relative expression of six of them was significantly higher (P ≤ 0.05) in parthenogenic embryos, while two genes showed non-significant (P ≥ 0.05) variation. The expression of BCL11A, PRP4B, TCP1, and TPI1 genes was significantly (P ≤ 0.05) DR in parthenotes in the micro-array study, while the TCP1 gene was up-regulated, and there was no variation in TPI1 gene expression in the PCR validation study. In conclusion, our findings demonstrate differential expression of a large number of genes in parthenotes at different stages of embryo development compared to fertilized embryos. Up-regulation of APOA1, MYBPC2, TCP1, and THRAP3 genes, suggest their crucial role in spontaneous facultative parthenogenic development in turkey birds.

The present study aimed to identify the effects of sugar and methods [slow freezing (SF) vs fast freezing (FF)] on post-thaw in-vitro functional characteristics of cryopreserved caprine SSCs (cSSCs) and the cells obtained from cryopreserved testis tissue of pre-pubertal Barbari bucks. For this, in experiment-1, cSSCs were isolated and cryopreserved by either SF or FF method with different nonpermeable [sugars; trehalose (140 mM; 140T or 400 mM; 400T), and sucrose (140 mM; 140S or 400 mM; 400S)] or/and permeable [5% ethylene glycol (EG) and DMSO] cryoprotectants. After one-wk of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment-2, the effectiveness of sugars [trehalose (140 mM) or sucrose (140 mM)] for cryopreservation of testicular tissues of pre-pubertal Barbari bucks using the SF or FF method was evaluated. After one-wk of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3-wk. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers expression. The recovery rate was 1.3, 1.3, and 1.1-folds higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (PGP9.5 and OCT-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue wt. was significantly (P

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4–5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90(+) cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p

The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Mesenchymal stem cells are now- a-days treated as the cornerstone of the cellular therapy and tissue engineering arena. These omnipresent cells can be isolated from a wide range of sources and can be differentiated and trans-differentiated into many cell types based on specific cellular command. In this study, we had isolated bone marrow derived mesenchymal stem cells from 1-2 years old apparently healthy goats. The heterologous cell population then subjected to magnetic activated cell sorting against CD73+, CD90+, CD105+, CD271+ and CD34- to exclude mesenchymal stem cells from haematopoietic stem cells. The cells were then cultured and incubated at 37 °C in humidified atmosphere with 5% CO2. After 3 passages the cells were tested for alkaline phosphatase activity. Furthermore, the cells were also evaluated based on their morphometric analysis.

Infectious diseases and aetiological agents related to female reproductive systems were extensively covered compared to its male counterpart. There needs a proper study to bridge this gap, where microflora and infectious agents of both male and female reproductive are mutually intelligible. With this study, we aimed to evaluate the microbial contamination of the preputial cavity and also screened for abortion-causing agents which are zoonotic as well. In goats, such types of abortions are caused by Brucella melitensis, Chlamydophila, Campylobacter and Coxiella etc. One of the major sources of contamination of semen is the preputial cavity, which is exposed to the external environment leading to spread of infection into the female via semen straws or by natural service. In the current study, good quality bucks (n = 32, Barbari = 12, Jamunapari = 10, Jakhrana = 10) which were routinely used for semen collection were screened for their preputial swabs, for the presence of the above pathogens. For detection of Brucella melitensis, OMP31 based TaqMan® probe real-time PCR assay was used, and for Chlamydia, 16srRNA gene based SYBR® green real-time PCR assay was employed for detection of Chlamydophila abortus. While for Campylobacter spp. and Coxiella burnetii, 16srRNA gene based conventional PCR and Trans-PCR were used, respectively. In the current study, of the screened preputial swabs, none of them showed positive for Brucella and Coxiella, but of the screened 32 samples 17 showed positive for Chlamydia (53.13%) and two (6.25%) showed positive for Campylobacter spp. The current study emphasizes on the farms and laboratories which were regularly involved in screening of brucellosis also often overlook the other potential non-brucella pathogens, causing abortions eventually incurring severe economic losses to the goat keepers.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4–6 months) and adult (~ 2 years) bucks during non-breeding (July–August; n = 4 each) and breeding (October–November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90(+) cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3(rd)-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p

Staphylococcus aureus is one of the most prevalent pathogens, and a causative agent of a variety of infections in humans and animals. A total of 48 semen samples were collected from healthy bucks of different breeds to investigate the prevalence of S. aureus . Antimicrobial resistance and virulence of the Staphylococcus isolates were determined to assess the adverse effects of them on buck fertility. The bacterial isolates were tentatively confirmed as Staphylococcus spp. based on the Gram’s staining, growth on Mannitol salt agar and catalase test. Overall, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds. Out of 36 staphylococcal isolates, 23 (47.92%) were coagulase negative (CoNS) and 13 (27.08%) were coagulase positive Staphylococcus (CoPS) based on the slide coagulase test. In the current study, on the basis of molecular characterization, we identified S. aureus , S. chromogenes, S. haemolyticus, S. sciuri, S. simulans and S. epidermidis amongst the staphylococcal isolates in the buck semen. This study revealed a high prevalence of Staphylococcus species in semen of the healthy bucks. The isolates exhibited varying degrees of multidrug resistance genotypically as well as phenotypically. The presence of antibiotic resistance and virulence genes may pose a potential threat to reproductive health of animals, highlighting the need for vigilant monitoring of these isolates at the time of semen cryopreservation.

Staphylococcus aureus is one of the most prevalent pathogens, and a causative agent of a variety of infections in humans and animals. Most studies concentrated on characterization of staphylococcus isolates and its antimicrobial resistance from various illness of veterinary importance, but there is no specific study that is available on isolates from reproductive tract of small ruminants and especially its semen. Hence, in the current study, a total of 48 semen samples were collected from healthy bucks of different breeds to investigate the occurrence of S. aureus. Antimicrobial resistance and virulence of the Staphylococcus isolates were determined to assess the adverse effects of them on buck fertility. The bacterial isolates were tentatively confirmed as Staphylococcus spp. based on the Gram's staining, growth on Mannitol salt agar and catalase test. Overall, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds and among them 23 (63.89%) were coagulase-negative (CoNS) and 13 (36.11%) were coagulase-positive Staphylococcus (CoPS) strains. The species identified by molecular characterization are S. aureus, S. chromogenes, S. haemolyticus, S. sciuri, S. simulans, and S. epidermidis from buck semen. Further, these isolates exhibited varying degrees of multidrug resistance genotypically as well as phenotypically. The presence of antibiotic resistance and virulence genes may pose a potential threat to reproductive health of animals, the animal handlers and livestock keepers, while simultaneously highlighting the need for vigilant monitoring of these isolates at the time of semen cryopreservation.

Objective: Buck reproductive health is the key for breeding and production of quality semen. To assess the health of breeding bucks, in this study, we detected the presence of Staphylococcus spp in semen. Staphylococcus aureus is a common commensal and opportunistic pathogenic bacteria and is also a cause of many diseases in animals. Besides this, it can also deteriorate the semen quality. Materials and Methods: In this study, we collected 48 semen ejaculates from healthy bucks of three breeds, namely, Jamunapari, Barbari, and Jakhrana to assess the presence of Staphylococcus spp. Besides bacteriological study, the semen was also assessed for semen quality parameters in infected as well as in non-infected semen samples. Results and Conclusion: The semen quality was significantly deteriorated with Staphylococcus infection. The bacterial infection was initially confirmed as Staphylococcus spp. based on the Gram’s staining and growth on Mannitol salt agar. Based on this preliminary bacteriological analysis, 52.08% (n = 25) of the samples were found positive for Staphylococcus spp. from the total 48 buck semen ejaculates belonging to three different goat breeds. The isolates were confirmed based on the basis of multiplex PCR and the species identified were S. aureus, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus chromogenes, Staphylococcus epidermidis, and Staphylococcus simulans directly in the buck semen. By this study, it is evident that semen can get contamination from a buck which has the presence of staphylococcus in the reproductive tract and semen quality is adversely affected. Hence, it is important to ensure the health and hygiene of the bucks maintained for semen production for artificial insemination.

The invasion of the male urogenital tract by microorganisms, and its subsequent effects on sperm fertilising ability, has not been well discussed in bucks. The present study was conducted to assess the bacterial load in fresh semen of the 2-6 years old bucks. For conducting the experiment, semen ejaculates from 18 bucks (6 from each breed namely Jakhrana, Jamunapari and Barbari) were used. We collected 5 ejaculates from each buck in each season (Summer-April to June, Rainy-July to Sept and Winter-November to January). Semen was collected with the artificial vagina (AV) method, and separate AV was used for each buck every time. The semen collection frequency was once in a week. Immediately after initial evaluation, collected semen samples were transferred to the microbiology laboratory of the institute. Thereafter, the semen samples were subjected to bacteriological examination to assess the microbial load. The results of the current study indicate that the microbial load in the semen was significantly (p

The growth and proliferation of mesenchymal stem cells are very sensitive in in vitro and a number of factors like media play a significant role in that context. In this study we assessed effect of different media on growth and proliferation of bone marrow derived mesenchymal stem cells (BMMSCs). The BMMSCs were isolated from caprine bone marrow and were subjected to magnetic activated cell sorting against CD90+, CD105+, CD271+and CD34- along with FC blocker. After characterisation, 2 × 104 cells were seeded in 12 well culture plates in four different media viz. MesenCult, MesenPRO, StemPro and complete DMEM (15 % FBS) to study their growth kinetic for 6 days from passage 0 (P0) to passage 3 (P3). The population doubling time (PDT) was derived from growth curve using logarithmic formula. The results showed that the BMMSCs growth and proliferation was highest in MesenCult media in P0 which varied significantly (p < 0.05) from rest of media and from P1 to P3, it was MesenPRO which yielded maximum cells (p < 0.05). The PDT was also in line with growth curve findings. In conclusion, the MesenPRO media had higher growth and proliferation rate from P1 to P3 although MesenCult had higher cell numbers in P0. In conclusion, the use of MesenPRO media could be a better option than conventional media when mesenchymal stem cells are used in clinical applications and other therapeutic purposes taking consideration to its higher growth and proliferation rate. And MesenCult would be a great option to harvest MSCs from P0.

The aim of the study was to explore the possibility of a better sugar suitable for storage of goat semen at refrigerated temperature. For this experiment, semen was collected from eight Jakhrana bucks maintained at Jakhrana unit, ICAR-CIRG, at twice a week interval using artificial vagina. Collected semen was preliminary evaluated, and better semen samples were pooled and divided into two parts. One part of the pooled semen was diluted in egg yolk, Tris, citric acid, and fructose diluter, whereas second part was diluted in egg yolk, Tris, citric acid, and glucose diluter. Then semen samples were kept in equilibration chamber for 4 h at 5 °C after proper dilution. Both the semen samples were evaluated for viability, motility, plasma membrane integrity, sperm abnormality, lipid peroxidation, acrosomal integrity, and capacitation status at 0 h, 24 h, 48 h, and 72 h after dilution. Significantly (P

Estrogen and its receptors are essential for sexual development and reproduction. Oestrogen receptor alpha (ERα) is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene ESR1 (oestrogen receptor1) responsible for better fertility. 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. The present study was undertaken to investigate the expression and presence of ESR1 gene in fertile and low-fertile male goat breeds. We identified ESR1 gene through various molecular tools. Genotyping was carried out by high resonance melting analysis using Roche Light Cycler 480(LC-480) system and found three different genotypes. Genotypic frequency-AA (blue-0.67), BB(Red-0.2), AB(Green-0.08) with allele frequency A(0.71 and B (0.29). The predominance of this gene in head of epididymis in fertile bucks was confirmed by SDS-PAGE, Western blotting and immunohistochemistry. From the results, we corroborated that the present study provides a useful and effective way to predict male fertility in goat breeds, which in turn increases the percentage of fertility in flock leading to more number of offspring in a kidding season.

This study was conducted to characterise pregnancy-associated glycoprotein (caPAG) in peripheral plasma during gestation and postpartum periods of nulliparous and multiparous does with one or two foetuses using a caPAG specific two-step sandwich ELISA system. Earliest time-points for detection of pregnancy and foetal number with appropriate cut-off values were identified. Plasma samples from 15 pregnant (multiparous: n = 8; nulliparous: n = 7; during pregnancy and postpartum period) and six non-pregnant (during oestrous cycle) goats were collected and analysed. Mean caPAG concentration was greater than the threshold for pregnancy detection (S-N = 0.40) on d22, peaked on d45 and remained unchanged until parturition. From d45 until parturition, caPAG concentration in multiparous does with two foetuses was 1.4 to 1.8 fold greater (P

The present study aimed to investigate whether follicular fluid, gonadotropins (FSH, LH and estradiol-17β), and combination of both types of supplementation (follicular fluid + gonadotropins) in maturation medium influence the transcript abundance of germ cell marker genes ( MATER , ZAR1 , GDF9 and BMP15 ) in goat oocytes. Grade I and II oocytes were collected from the goat ovaries and matured further under in-vitro conditions using four different maturation regimens viz , group B containing basal media (TCM 199 + BSA + serum), group C (basal media + 10% follicular fluid), group D (basal media + FSH + LH + estradiol-17β) and group E (basal media + 10% follicular fluid + FSH + LH + estradiol-17β). Group A consisted of immature oocytes. Expression profile of MATER, ZAR1, GDF9, and BMP15 was analyzed in matured oocytes of different groups as well as immature oocytes using real-time PCR. The relative abundance of MATER gene in immature oocytes (group A) was found to be significantly higher (p ZAR1 was found to be significant different (P GDF9 and BMP15 was significantly higher (P MATER and ZAR1 transcript were down regulated after maturation; however BMP15 and GDF9 transcripts were upregulated after maturation. For better in vitro embryo production (IVEP) out comes goat oocytes may be matured in vitro in TCM-199 supplemented with follicular fluid and gonadotropins.

Temperature Humidity Index (THI) is a single value representing the combination of air temperature and humidity associated with the level of thermal stress. Ejaculates (n = 890) from 12 Barbari bucks (2–4 years) maintained under semi-intensive management system were utilized to find out the effect of THI on sexual behavior and semen quality in Barbari buck. Semen with mass activity ≥ 4 was considered freezable. Buck mounting per cent during May and June were high whereas comparatively lower during November and December. Interestingly freezable quality of semen was good during November and December. The poorest freezable quality was observed during January and February. The comfortable THI (

This study was designed to investigate plasma profile of pregnancy-associated glycoprotein (PAG), progesterone (P4) and estrone sulfate (E1S) during early- and mid-pregnancy. The goal was to explore the relationships with values for reproductive variables, to detect the most reliable predictor variable, and to identify the most desirable time point for blood collection for determining fetal number in goats. After ultrasonographic examination at d35–40 post-mating, blood sampling of 15 pregnant goats (total 18) was continued until d114. The PAG profile was characterized by gradual increase during early pregnancy from d26 to d51 and thereafter concentrations were relatively constant until d114 of gestation. The effect of fetal number on plasma PAG, P4 and E1S was first evident on d28, d51 and d26, respectively. During mid-pregnancy, does with twins had a greater (P

Thirty six ejaculates from 6 adult Barbari bucks (2–4 years old) maintained at C.I.R.G under semi intensive management system were used to find out the freezability of buck semen at different levels of vitamin C (0.0 μM, 45.42 μM, 56.78 μM, 68.13 μM) by conventional method of freezing. The ejaculates were collected twice at weekly intervals by artificial vagina. The semen samples were diluted with tris–citric acid fructose diluents having 10% (v/v) egg yolk and 6% (v/v) glycerol as cry protectant agent. The semen samples were extended to maintain sperm concentration approximately 100–120 million per dose. Filling and sealing of straws were done at 5 °C in cold handling cabinet after 4 h of equilibration period, then straws were vapour frozen for 10 min above 2 cm of liquid nitrogen and finally stored into liquid nitrogen container. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling test were conducted to check the freezability. Post thaw motility, live sperm count, acrosomal intergrity and hypo osmotic swelling positive spermatozoa differed significantly among groups and they were the highest in 1% group. The result indicated that vitamin C at the level of 56.78 μM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen.

A study was aimed to optimize the electrofusion parameters for generation of caprine tetraploid in vitro fertilized embryos. Freshly recovered 2-cell goat in vitro fertilized embryos were submitted to electrofusion with varying voltages (1.0, 1.2 and 1.4 kV/cm) and time duration (2, 4 and 6 μ sec) combinations to produce tetraploid goat embryos. Fused embryos were cultured in RVCL (Research vitro cleave) media further for 8 to 12 days for subsequent embryo development. Fusion, cleavage and blastocyst rate at voltage 1.2kV/cm for 4 μ sec duration was significantly higher (P

The objective of this study was to compare the effectiveness of different activation treatments of in vitro matured oocytes and their developmental potency in potassium simplex optimization medium. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulphate per ml. A total of 1004 cumulus oocyte complexes (COC's) were collected from 454 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (10 μg/ml), oestradiol-17β (1 μg/ml) supplemented with 10% fetal bovine serum and 3 mg/ml BSA at 38.5 °C and 5% CO2 in an incubator under humidified air for 27 h. After 27 h of IVM, oocytes were denuded, washed and selected 933 in vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n = 579), were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in KSOM. Group 2 in vitro matured oocytes (n = 145) were exposed to 7% ethanol for 5 min followed by treatment with 10 μg/ml CHX for 4 h in KSOM. Group 3 in vitro matured oocytes (n = 100) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 μg/ml CHX for 4 h in KSOM. Group 4 in vitro matured oocytes (n = 109) were cultured for 4 h without any chemical activation treatment in KSOM medium (control). After 4 h of culture in different chemicals, the oocytes were washed five to ten times in the culture medium (KSOM) and cultured in 50 μl drops of KSOM. Development of activated oocytes was observed at every 48 h till day 10 post activation under an inverted phase contrast microscope (200x, Nikon, Japan). The cleavage rate in groups 1, 2, 3 and 4 were 42.83%, 58.62%, 74.0% and 0.00%, respectively and morula production in groups 1, 2 and 3 were 24.59%, 30.58% and 31.08%, respectively. These results indicated that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 μg/ml CHX for 4 h in KSOM is most favorable for parthenogenetic caprine embryos production.

The objective of this study was to compare the effectiveness of different concentrations of ethanol treatment for activation of oocytes and their developmental potency in vitro. Ovaries were collected from a local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulphate per ml. A total of 2680 cumulus oocyte complexes (COCs) were collected from 899 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (10 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO 2 in an incubator under humidified air for 27 h. After 27 h of IVM, oocytes were denuded, washed and randomly divided into five groups. Group 1 consisted of in vitro matured oocytes ( n = 403) as control which were washed with KSOM medium without ethanol. Group 2 consisted of in vitro matured oocytes ( n = 412) activated with 1% ethanol for 5 min in KSOM medium. Group 3 was comprised of in vitro matured oocytes ( n = 362), activated with 1% ethanol for 5 min in KSOM medium. Group 4 was comprised of in vitro matured oocytes ( n = 564) activated with 5% ethanol for 5 min in KSOM medium. Group 5 consisted of in vitro matured oocytes ( n = 634) activated with 7% ethanol for 5 min in KSOM medium. Group 6 consisted of in vitro matured oocytes ( n = 305) activated with 9% ethanol for 5 min in KSOM medium. After 5 min activation, the oocytes were washed 5–10 times in the culture medium (KSOM) and cultured in 50 μl drop of KSOM. Development of activated oocytes was observed at every 24 h till day 10 post insemination under inverted phase contrast microscope (200×, Nikon, Japan). The percentage of cleavage and morula production in groups 1, 2, 3, 4, 5 and 6 were 0.00% and 0.00%, 0.00% and 0.00%, 8.28% and 6.66%, 10.43% and 26.31%, 33.19% and 29.26%, 40.32% and 14.63%, respectively. These results suggested that the activation of in vitro matured oocytes by 7% ethanol for 5 min in KSOM is most favorable for parthenogenetic caprine embryos production.

Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occurs without fertilization by a male. Parthenogenesis occurs naturally in aphids, Daphnia, rotifers, nematodes and some other invertebrates but can also be induced efficiently in mammalian oocytes by providing appropriate stimuli invitro. Recently, parthenogenesis has attracted wide attention because of the role of activated oocytes in the field of research that have been described such as intra cytoplasmic sperm injection, cloning by nuclear transfer, somatic cell cloning, investigating culture conditions etc. & potential for deriving pluripotent stem cell lines and their differentiation into various cell lines that can be utilized for various tissue engineering applications. The parthenogenetically activated oocytes possess maternal genome and can developed in to either haploid, diploid or polyploidy embryos with the help of it we can analyze the possible role of all the genes involved in imprinting processes as well as the role the paternal genome plays during early embryo development by comparing them with fertilized embryos. Several methods are able to induce parthenogenetic activation through the elevation of cytoplasmic free calcium in oocytes. But one common, universal method or activation agents has not been developed for all species because the process is highly specific for each species. Therefore, activation step for each species need to be optimized accordingly. This review describes the general method of activation of mammalian oocytes and their genomic imprinting analysis.

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.

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO2 in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes.

The aim of the study was to evaluate the optimum concentration of the estrous goat serum for goat in vitro oocyte maturation. Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 °C), supplemented with 100 IU penicillin G and 100 μg streptomycin sulfate/ml. Cumulus-oocyte complexes (COC's) were obtained by puncture of 1–4 mm diameter follicles with a 18G needle attached to a disposable syringe, washed 10 times in TCM-199 hepes modification with Earl's salts and supplemented with 50 μg/ml gentamycin, 100 μg/ml l -glutamine and 0.25 mM sodium pyruvate without any serum supplementation. The COC's were randomly divided into four groups. Group 1 (n = 105) COC's were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 105) COC's were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 (n = 110) COC's were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 (n = 112) COC's were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. After 24–27 h of IVM, oocytes were denuded with the aid of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p

A study was conducted on 18 healthy Jakhrana goats, maintained under semi-intensive management system at ICAR-Central Institute for Research on Goats, Mathura (UP). Serum testosterone levels in males and progesterone in females were studied during three reproductive stages (pre-pubertal, pubertal and sexually mature) in 12 goats. In addition, serum progesterone levels were determined during pregnancy and on the day of kidding in 6 does. Blood samples from goats (6 of each sex) at 10–12 months (pre-pubertal), 13–14 months (pubertal) and 15–16 months of age (sexually mature) were collected at fortnightly intervals. Similarly blood samples from six pregnant does were collected at 30, 60, 90, 120 days of pregnancy and on the day of kidding. The mean serum testosterone levels in male goats during the pre-pubertal, pubertal and sexually mature stages were 2.55±0.31, 2.80±0.21 and 4.11±0.14ng/ml, respectively and differed significantly (P 0.05) between stages. Progesterone level remained elevated and almost identical during pregnancy with minor fluctuations at different stages of pregnancy (5.04±0.25, 5.21±0.22, 5.01±0.24 and 4.78±0.14 ng/ml, respectively). Its level then sharply declined on the day of kidding (0.67±0.14 ng/ml). Progesterone level on the day of kidding was significantly lower (P

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.

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

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%).