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Background: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility., Objective: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1., Materials and Methods: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×10 8 mL -1 . Post-cryopreserved sperm were assessed for motility and a range of other functional parameters., Results: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%)., Conclusion: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications., (© 2023. Springer Nature Limited.)

The objective of the present study was to establish a workable approach for the production of germ cell (GC)-depleted recipient goat model using intra-testicular busulfan treatment and transplantation of cultured and enriched caprine-male GC (cmGCs) into the homologous recipients under ultrasonography (USG) guidance. The evaluation of post-transplantation colonization of donor cmGCs and restoration of the normal architecture of seminiferous tubules (ST) was performed. For this, the cmGCs of pre-pubertal male goats were isolated and enriched by differential platting for culture until the third passage. Thereafter, cells were harvested and further enriched by magnetic-activated cell sorting using rabbit-anti-CD90 antibody. After confirmation of metabolic viability (MTT-assay) and cluster-forming ability (crystal violet staining) of CD90+ cmGCs, the cells were labeled with a lipophilic red-fluorescent dye (PKH26) before transplanted into the recipient male goats by injection directly into the mediastinum testes under USG guidance. The colonization and repopulation of transplanted CD90+ cmGCs into the recipient ST was observed up to 8 weeks post-transplantation. The PKH26-labeled donor cell-derived colonies were identified in enzymatically digested ST and cryosections of recipient testes. Moreover, histochemical analyses revealed the restoration of the normal architecture of ST of recipient testis after GC transplantation. Therefore, the results suggest that the reproductive competence of infertile animals can be restored through mGC therapy and thus the methodology presented herein could be useful to obtain donor mGCs-derived functional male gametes in the recipient animal testis., (© 2022 S. Karger AG, Basel.)

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 spermatogonial stem cells (cSSCs) and the cells obtained from cryopreserved testis tissue of prepubertal Barbari bucks. For this, in experiment 1, cSSCs were isolated and cryopreserved by either SF or FF method with different non-permeable (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 dimethyl sulfoxide) cryoprotectants. After 1 week 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 prepubertal Barbari bucks using the SF or FF method was evaluated. After 1 week of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3 weeks. 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-fold higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (protein gene product 9.5 and octamer-binding transcription factor-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue weight was significantly (p &lt; 0.05) higher when cryopreserved using 140 mm trehalose compared with other groups. The results of immunocytochemical analyses imply the expression of pluripotent stem cell markers in cSSCs following cryopreservation. Overall, the outcome of the study demonstrates different effects of sugars and methods on post-thaw functional properties of cSSCs with superiority of 140 mm trehalose using SF method over other treatment groups. These results are important for ex vivo expansion and differentiation of cSSCs for fertility preservation and their other downstream applications., (© 2023 S. Karger AG, Basel.)

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and β-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the research report., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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  < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly ( p  < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and β1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs., Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-021-00515-x., Competing Interests: Conflict of interestThe authors declare that no conflict of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (β1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs., (© 2021. Cell Stress Society International.)

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O 2 ) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O 2 ). The comparative information about the effects of low and normal O 2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O 2 ) and normoxia (21% O 2 ). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O 2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Defensins are antimicrobial peptides and uniformly spans the entire sperm surface and is not exclusive to a specific domain. Goat β-defensin-1 helps in initiation of motility and capacitation of sperm., Objective: To know the status of β-defensin-1 in blood, semen and its effect on post thaw fertility gene expression in Indian goat breeds., Materials and Methods: Semen was extended and divided for estimation of β-defensin-1 and cryopreserved having different concentrations of β-defensin-1., Results: Bet defensin-1 concentration (pg/mL) in neat semen, sperm pellet and seminal plasma was significantly higher (P< 0.05) in goat breed Barbari followed by Jamunapari and Jakhrana. β-defensing-1 was also high in Jakhrana blood followed by Barbari and Jamunapari. The post thaw motility, live sperm, acrosome intactness and hypo osmotic swelled sperms were significantly higher (P< 0.05) with 10 ng/mL β-defensin in the semen dilutor., Conclusion: Beta defensin (10 ng/mL) in semen dilutor may be used as immuno-modulator to get better post thaw quality suitable 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 < 0.05) higher in the Jamunapari bucks and in aged bucks. Bacteriospermia in different seasons was not significantly varied; however, nonsignificant increase in microbial load during the rainy season was observed. Overall, the average bacterial load in the semen of Jamunapari, Barbari and Jakhrana bucks was found 540.50 ± 55.88 CFU/ml, 391.81 ± 46.33CFU/ml and 388.93 ± 44.71 CFU/ml respectively. No significant difference in bacterial counts in the subsequent ejaculates among bucks was observed. Moreover, correlation analysis revealed that the proportions of motility, viability, plasma membrane integrity and acrosomal integrity were negatively influenced by the increased bacterial contamination of buck semen., (© 2021 Wiley-VCH GmbH.)

Objective: This study was conducted to assess the level of pregnancy-associated glycoprotein (PAG) in whole and skim milk samples, and its suitability for early pregnancy diagnosis in goats., Methods: A two-step sandwich enzyme-linked immunosorbent assay (ELISA) system for estimation of milk PAG was developed and validated, which employed caprine-PAG specific polyclonal antisera. Whole and skim milk samples (n = 210 each) from fifteen multiparous goats were collected on alternate days from d 10 to d 30, and thereafter weekly till d 51 postmating. PAG levels in milk samples were estimated by ELISA and the pregnancies were confirmed at d40 post-mating by transrectal ultrasonography (TRUS)., Results: The level of PAG in whole and skim milk samples of both pregnant and nonpregnant goats remained below the threshold values until d 24 after mating. Thereafter, PAG concentration in whole and skim milk increased steadily in pregnant goats, whereas it continued below the threshold in non-pregnant does. The PAG profiles in whole and skim milk of pregnant goats were almost similar and exhibited strong positive relationship (r = 0.891; p<0.001). Day 26 post-mating was identified as the first time-point for significantly (p<0.05) higher milk PAG concentration in pregnant goats than to non-pregnant goats. When compared to TRUS examination for pregnancy diagnosis, the accuracy and specificity of PAG ELISA using whole and skim milk samples were 94.5% and 95.4%; and 95.3% and 100%, respectively. The high values of area-under-curve (0.904 [whole milk] and 0.922 [skim milk]), demonstrate outstanding discrimination ability of the milk assays. Among the sampling dates chosen, d 37 post-mating was identified as the best suitable time point for collection of milk samples to detect pregnancy in goats., Conclusion: The PAG concentration in whole and skim milk of goats collected between days 26 and 51 post-breeding can be used for the accurate prediction of pregnancy and may be useful for assisting management decisions in goat flocks.

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 × 10 4 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2020 Wiley-VCH GmbH.)

Objective: The aim of the study was to explore the possibility of a better sugar suitable for storage of goat semen at refrigerated temperature., Materials and Method: 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., Results: Significantly (P < 0.05) higher motility was observed at 24 h in extender containing glucose as compared with extender containing fructose but motility was decreased at 48 h and 72 h. Number of capacitated sperm increased significantly (P < 0.05) and acrosomal integrity was decreased significantly (P < 0.05) at 72 h in extender containing glucose. The other parameters like viability and plasma membrane integrity were decreased significantly (P < 0.05) at 72 h and lipid peroxidation as well as sperm abnormality increased significantly (P < 0.05) in extender containing glucose., Conclusion: From this study, it can be concluded that fructose is better diluent sugar for refrigerated storage of buck semen.

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 < 0.001) than those with one foetus. For the ELISA, 0.83 (S-N) was the most appropriate cut-off to differentiate does with two from those with a single foetus with an overall sensitivity and accuracy of 88.9% and 84.7%, respectively. Circulating caPAG concentration in multiparous goats was greater (P < 0.05) compared with nulliparous goats during the early pregnancy and postpartum periods. After parturition, caPAG concentrations markedly decreased and were basal within 14 days postpartum. In conclusion, using the caPAG specific ELISA, results indicated there were unique gestational and postpartum profiles for caPAG concentrations that are affected by number of foetuses and parity of the doe. The marked decrease in concentration of caPAG following parturition indicates there would not be compromising of the detection of subsequent pregnancies in goats using this technique., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study was designed to investigate plasma profile of pregnancy-associated glycoprotein (PAG), progesterone (P4) and estrone sulfate (E 1 S) 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 E 1 S was first evident on d28, d51 and d26, respectively. During mid-pregnancy, does with twins had a greater (P < 0.05) PAG (S-N = 2.54 ± 0.12 compared with 1.59 ± 0.11), P4 (18.91 ± 0.67 compared with 14.51 ± 0.47 ng/mL) and E 1 S (16.34 ± 0.76 compared with 11.32 ± 0.44 ng/dL) as compared with does with a singleton fetus. Plasma PAG but not P4 and E 1 S was positively correlated with fetal number and birth weight of kids during early pregnancy. Multivariate linear regression and discriminant function analyzes allowed for identification of plasma PAG as the most reliable predictor for fetal number and birth weight of kids. Furthermore, d58 was the most suitable single time point for prediction of fetal number using PAG as a biomarker. In conclusion, plasma profile of PAG, P4 and E 1 S was affected by fetal count. Plasma PAG was identified as the most reliable predictor variable of fetal number and birth weight of kids as compared to plasma P4 and E 1 S., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The present study was undertaken to assess the effect of different chemical activators along with 6-DMAP on in vitro matured caprine oocytes. From 4332 ovaries, 14235 cumulus oocyte complexes (COCs) were collected which were matured in TCM-199 medium containing follicle stimulating hormone (FSH) (5 µg/ml), Leutinizing hormone (LH) (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% fetal bovine serum, 10% follicular fluid and 3 mg/ml bovine serum albumin (BSA) at 38.5°C and 5% CO 2 in an incubator under humidified air for 27 h. In group 1 (control), 3117 in vitro matured oocytes were co incubated with sperms for 18 h in ferttalp medium. In group 2, 3563 in vitro matured oocytes were activated with 7% ethanol for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR 2 aa medium. In group 3, 3109 in vitro matured oocytes were activated with 5 μM ionomycin for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR 2 aa medium. In group 4, 3455 in vitro matured oocytes were activated with 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR 2 aa medium. Oocytes were cultured in 50 µL drops of research vitro cleave (RVCL) medium for embryo development. The cleavage rate, morula and blastocyst production in group 1, 2, 3 and 4 were 26.07 ± 2.37%, 14.91 ± 2.91 & 1.45 ± 0.71%, 49.57 ± 3.79%, 20.07 ± 2.38% & 5.29 ± 1.42%, 50.18 ± 3.59%, 15.26 ± 2.87% & 1.85 ± 0.72% and 80.26 ± 2.30%, 35.33 ± 2.67 & 7.10 ± 0.89%, respectively. These results indicated that the activation of in vitro matured oocytes by 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h is most favorable for parthenogenetic caprine embryos production., Competing Interests: None of the authors have any conflict of interest to declare.

The aim of the present investigation was to compare Tyrode's albumin lactate pyruvate (TALP) medium and TRIS citrate medium for capacitation of caprine sperm. In experiment 1 capacitation was assessed by chlortetracycline assay and in experiment 2 with in vitro fertilization and embryo development. In experiment 2, cumulus oocyte complexes (COCs) recovered by slicing the caprine ovaries were matured in maturation medium for 27 h in humidified atmosphere at 38.5°C with 5% CO 2 . After 27 h of culture a total of 2480 in vitro matured oocytes were selected and randomly divided into two groups. Group 1 (n=1124) matured oocytes were fertilized by the spermatozoa capacitated in TALP medium and in group 2 (n=1356) matured oocytes were fertilized by the spermatozoa capacitated in TRIS citrate medium. The results of experiment 1 indicated a comparatively more number of sperms with Chlortetracycline (CTC) Pattern B in TRIS citrate than TALP medium (55.32 ± 0.91% vs 47.96 ± 0.20%). In experiment 2, the cleavage rate and blastocyst production were higher following capacitation of spermatozoa in TRIS citrate than TALP medium. In conclusion, TRIS citrate can be used as an alternative and effective media for sperm capacitation to get higher cleavage rate and blastocyst production in goat., Competing Interests: On behalf of all co-authors, the corresponding author indicates that there is no conflict of interest involved in publishing this research paper.

The present study was undertaken to analyze the expression pattern of estrogen receptor 1 gene (ESR1) in Barbari bucks (fertile and non-fertile) identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the spleen by Trizol method. The expression pattern of ESR1 gene was analyzed using real time polymerase chain reaction (RT-PCR). The expression pattern of ESR1 gene was analyzed by RT-PCR (Roche LC-480). Relative quantification by RT-PCR indicated that the ESR1 gene expression showed more fold in fertile bucks as compared to non-fertile.

Aim: The objective of the study was to determine the effect of the management system (intensive and semi-intensive) and season (autumn and winter) on semen freezability in Jakhrana bucks., Materials and Methods: A total of 24 Jakhrana bucks of same body weight and age (BW=30 kg, age=1 year) were randomly allotted into two groups, viz., Group I (intensive system, 12 bucks) and Group II (semi-intensive system, 12 bucks). These two groups were statistically tested for their homogeneity with respect to age and BW. Semen was collected twice weekly using an artificial vagina during two seasons: autumn (September-November) and winter (December-February). A total of 240 semen samples (120 from each group and season) were evaluated for post-thaw motility (PTM), viability, abnormality, functional membrane integrity (hypo-osmotic swelling [HOS]) response and acrosomal integrity., Results: The mean values of PTM and acrosomal integrity of spermatozoa were significantly (p<0.01) higher in Group II as compared to Group I. The mean values of viability and abnormality were also differed significant (p<0.05) between groups. However, the mean values of HOS response were found non-significant (p>0.05) between groups. The season showed a significant effect on all parameters except viability and HOS response. The PTM and acrosomal integrity of spermatozoa were significantly (p<0.01) higher in winter as compared to autumn season. Abnormality of spermatozoa was significantly (p<0.05) lower in winter season., Conclusions: This study indicates that both management system and season influence semen freezability. The semen collected from bucks reared under the semi-intensive system and winter season showed better semen freezability characteristics.

This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR2aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes (COC's) were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=226) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Group 2 in vitro matured oocytes (n=294) were exposed to 7% ethanol for 5 min followed by treatment with 10 µg/ml CHX for 4 h in mCR2aa medium. Group 3 in vitro matured oocytes (n=325) 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 mCR2aa medium. Group 4 in vitro matured oocytes (n=108) were cultured for 4 h without any chemical treatment in mCR2aa medium (control). The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa is most favorable for parthenogenetic caprine embryos production.

The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC's) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF.

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.

The aim of this study was to investigate the effect of treating repeat breeder dairy crossbred cows with different doses of GnRH analogue through i.m. at the time of artificial insemination, on pregnancy rates from their first service after treatment and overall pregnancy rates. One hundred and thirty seven crossbred dairy cows with a history of repeat breeding and eligible after 6-8 infertile services but clinically free of diseases were selected for the study. The animals were randomly divided into three groups. Group 1 (n = 55) cows were treated intramuscularly with each 20 microg Buserelin-acetate (Receptal, Hoechst Roussel Vet GmbH) at the time of artificial insemination. Group 2 (n = 40) cows were treated intramuscularly with each 10 microg Buserelin-acetate at the time of artificial insemination. Group 3 (n = 42) cows were treated intramuscularly with saline as control at the time of artificial insemination. The first service pregnancy rates in Groups 1-3 were 45, 25 and 17%, respectively. Similarly, the overall conception rates in Groups 1-3 were 87, 58 and 48%, respectively. The results indicated that the pregnancy rate in crossbred cows could be improved by the GnRH treatment. The higher dose of GnRH significantly increased (P < 0.05) the first service as well as overall pregnancy rate in a dose dependent manner in repeat breeder crossbred cow bred previously 6-8 times unsuccessfully., ((c)2006 Elsevier B.V. All rights reserved.)