Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

BACKGROUND: Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. METHODS AND RESULTS: Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures [38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. CONCLUSION: It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

l-Ergothioneine improves the developmental potential of in vitro sheep embryos without influencing OCTN1-mediated cross-membrane transcript expression.

SummaryThe objective of the study was to investigate the effect of l-ergothioneine (l-erg) (5 mM or 10 mM) supplementation in maturation medium on the developmental potential and OCTN1-dependant l-erg-mediated (10 mM) change in mRNA abundance of apoptotic (Bcl2, Bax, Casp3 and PCNA) and antioxidant (GPx, SOD1, SOD2 and CAT) genes in sheep oocytes and developmental stages of embryos produced in vitro. Oocytes matured with l-erg (10 mM) reduced their embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH in matured oocytes that in turn improved developmental potential, resulting in significantly (P < 0.05) higher percentages of cleavage (53.72% vs 38.86, 46.56%), morulae (34.36% vs 20.62, 25.84%) and blastocysts (14.83% vs 6.98, 9.26%) compared with other lower concentrations (0 mM and 5 mM) of l-erg without change in maturation rate. l-Erg (10 mM) treatment did not influence the mRNA abundance of the majority of apoptotic and antioxidant genes studied in the matured oocytes and developmental stages of embryo. A gene expression study found that the SLC22A4 gene that encodes OCTN1, an integral membrane protein and specific transporter of l-erg was not expressed in oocytes and developmental stages of embryos. Therefore it was concluded from the study that although there was improvement in the developmental potential of sheep embryos by l-erg supplementation in maturation medium, there was no change in the expression of the majority of the genes studied due to the absence of the SLC22A4 gene in oocytes and embryos that encode OCTN1, which is responsible for transportation of l-erg across the membrane to alter gene expression.

Molecular cloning and expression of FGF2 gene in pre-implantation developmental stages of in vitro-produced sheep embryos.

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor-2 (FGF-2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non-pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2 , 95% humidity for 22-24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6-7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real-time polymerase chain reaction using sequence-specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2-cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre-implantation stages of embryo. It can be concluded that FGF-2 plays a significant role in pre-implantation and early development of embryos in sheep.

Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L-carnitine.

The objective of this study was to find out the impact of L-carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post-fertilization medium separately. Subsequent objective was to observe the L-carnitine-mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L-carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L-carnitine during post-fertilization period. So it is suggested to use L-carnitine during maturation than post-fertilization period. Antiapoptotic and proliferative effects of L-carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L-carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but supplementation of L-carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control. L-carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L-carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16-cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L-carnitine supplementation. In conclusion, L-carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L-carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.

L-carnitine Mediated Reduction in Oxidative Stress and Alteration in Transcript Level of Antioxidant Enzymes in Sheep Embryos Produced In Vitro.

The objective of this study was to find out the effect of L-carnitine on oocyte maturation and subsequent embryo development, with L-carnitine-mediated alteration if any in transcript level of antioxidant enzymes (GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2) in oocytes and developing sheep embryos produced in vitro. Different concentrations of L-carnitine (0 mm, 2.5 mm, 5 mm, 7.5 mm and 10 mm) were used in maturation medium. Oocytes matured with 10 mm L-carnitine showed significantly (p < 0.05) higher cleavage (66.80% vs 39.66, 41.76, 44.64, 64.31%), morula (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst (33.22% vs 7.66, 9.19, 10.71, 28.57%) percentage as compared to lower concentrations (0 mm, 2.5 mm, 5 mm and 7.5 mm). Cleavage percentage between 10 mm and 7.5 mm L-carnitine were not significantly different. Maturation rate was not influenced by supplementation of any experimental concentration of L-carnitine. There was a significant (p < 0.05) decrease in intracellular ROS and increase in intracellular GSH in 10 mm L-carnitine-treated oocytes and embryos than control group. Antioxidant effect of L-carnitine was proved by culturing oocytes and embryos with H2O2 in the presence of L-carnitine which could be able to protect oocytes and embryos from H2O2-induced oxidative damage. L-carnitine supplementation significantly (p < 0.05) upregulated the expression of GPx and downregulated the expression of SOD2 genes, whereas the expression pattern of SOD1 and GAPDH (housekeeping gene) genes was unaffected in oocytes and embryos. It was concluded from the study that L-carnitine supplementation during in vitro maturation reduces oxidative stress-induced embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH that in turn improved developmental potential of oocytes and embryos and alters transcript level of antioxidant enzymes.

Maturation timing and fetal bovine serum concentration for developmental potential of sheep oocytes in vitro.

The success of in vitro embryo production (IVEP) in animals has improved over time, employing a variety of culture media. Here, we assessed the maturation timing and developmental potential of sheep oocytes in vitro at different concentrations of fetal bovine serum (FBS): Cumulus oocyte complexes (COCs) were aspirated from follicles (2-6 mm) of sheep ovaries collected from local slaughter house. COCs were randomly divided into two groups and matured at 38.5'C, 5% CO2 for 24 h (Group I) and 27 h (Group II). Oocytes cultured for 27 h showed significantly (P <0.05) more maturation than those cultured for 24 h (82 vs. 76%) followed by more cleavage (35 vs. 30%), morula (53 vs. 39%) and blastocyst (17 vs. 11%) percentage. In the second experiment, oocytes were randomly divided into two groups and matured with 10% FBS (Group I) and 20% FBS (Group II) for 27 h supplemented with pyruvate, glutamine, LH, FSH and estradiol. After maturation, oocytes were fertilized by fresh semen for 18 h. Presumptive zygotes in both the groups were again divided into two groups and culturedin 10 and 20% FBS during post fertilization period, respectively. Different FBS concentration in maturation medium did not influence maturation percentage (82 vs. 79%) significantly. Out of culture groups, presumptive zygotes matured in 20% FBS and cultured in 20% FBS during post fertilization period showed significant increase in cleavage percentage (44 vs. 39, 35 and 27%) as compared to other groups but subsequent development to morula (55 vs. 53, 43 and 40%) and blastocyst (20 vs. 17, 16 and 15%) percentage were more in the group matured in 10% FBS and cultured in 20% FBS during post fertilization period.

Isolation and characterization of luteal cells in buffalo (Bubalus bubalis).

The objective of the study was to evaluate the morphological and functional characteristics of luteal cells isolated from buffalo ovary. Luteal cells exhibited columnar morphology and contact inhibition at the stage of confluence. Protein concentrations increased linearly from Day 3 to Day 7 of culture. DNA concentrations increased from Day 3 to Day 5 and then declined to Day 7 of culture. PGF2alpha concentrations decreased progressively from Day 3 to Day 7 of culture. It was concluded that buffalo luteal cells could serve as an excellent model for studying the specific role of PGF2alpha in maternal recognition of pregnancy and implantation.

Suppression of COX-2 mRNA abundance in in vitro cultured goat (Capra hircus) endometrial cells by RNA interference and effect on PGF2-α and PGE2 concentrations.

Cyclooxygenase-2 (COX-2) has important functions in the synthesis and release of endometrial prostaglandin F2α (PGF2α). Excessive production of COX-2 leads to an increase in endometrial PGF2α synthesis and subsequently causes luteolysis and early embryonic mortality. The aim of this study was to investigate in goats the effects of COX-2 small interference RNA (siRNA) on COX-2 mRNA abundance and the secretion of PGF2α and PGE2 in goat endometrial cells. Endometrial cells isolated from goat uteri were cultured at 38.5 °C and 5% CO2. The cells were treated with different concentrations (0, 10, 25, 50, 100, 250, 500, 750 and 1000 nM per well) of three different COX-2 siRNAs at confluency for 24 h. At 24 h post culture, COX-2 mRNA abundance was quantified using qPCR and PGF2α and PGE2 concentrations were quantified in the culture medium. There was a lesser relative abundance of COX-2 mRNA in endometrial cells at 100 to 1000 nM siRNA. The greatest extent of abundance suppression, however, was observed with 1000 nM siRNA. Transfection of COX-2 siRNA (1000 nM) to endometrial cells suppressed the COX-2 mRNA abundance by 77%, 82%, and 84% with siRNA 1, 2, 3, respectively. Furthermore, with COX-2 siRNA transfected cells, there was a lesser (P < 0.05) PGF2α concentration than in cells not transfected, whereas PGE2 secretion was not affected. The results of the study provide evidence that COX-2 siRNA used in this study suppresses COX-2 mRNA abundance and PGF2α secretion but there was no association between PGE2 concentrations and COX-2 mRNA abundance in goat endometrial epithelial cells.

In Vitro Embryo Production in Sheep.

The recent advances in biotechnological research have led to development of many advanced reproductive techniques and biological tools which are set to revolutionize the productive efficiency of livestock species. The development of technology for sequencing of whole genomes and mass screening of gene regulation has widened our approach to genetic profiling and mapping, as well as furthering our understanding of underlying physiological mechanisms. The newer biotechnologies of gene transfer, in vitro fertilization and embryo production, cloning, and stem cell technology have been developed and are being refined with efficiencies suitable for use in animal farming. Efficient in vitro systems for maturing oocytes, fertilizing, and developing embryos have resulted in commercial in vitro production of embryos. Here we describe in vitro maturation, in vitro fertilization, embryo production, embryo culture, and quantitation of gene expression in sheep embryos.

Relationship of organic mineral supplementation and spermatozoa/white blood cells mRNA in goats.

The antioxidant properties and the protective role of organic zinc (Zn) and copper (Cu) in white blood cells (WBCs) and spermatozoa were analyzed through quantification of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase 4 (GPx4) and nuclear factor erythroid 2-like 2 (NFE2L2) and correlations were determined with sperm functional characteristics in Osmanabadi bucks. Bucks (aged 5 months; n = 40) were divided into ten groups, and the dietary treatments comprised of a control and nine treatment groups as follows: organic Zn as Zn 20, Zn 40 and Zn 60, organic Cu as Cu 12.5, Cu 25, Cu 37.5 and combined organic Zn and Cu as Zn 20+Cu 12.5, Zn 40+Cu 25, Zn 60+Cu 37.5, respectively per kg dry matter for a period of 8 months. The blood (120 and 240 days) and semen (240 days: 40 × 4 = 160) samples were collected from 40 bucks. In WBCs: the relative abundance of mRNA for SOD1, CAT, GPx4, NFE2L2 was greater (P < 0.05) in (120 and 240 days) in majority of the mineral supplemented animals. In spermatozoa: the relative abundance of SOD1, NFE2L2, GPx4 and CAT mRNA was greater (P < 0.05) in selected treatment groups. The abundance of SOD1 mRNA in WBCs was positively correlated (P < 0.05) with sperm mass motility (r = 0.692, P = 0.027). The abundance of GPx4 mRNA was negatively correlated (P < 0.05) with type A sperm (straightness; STR) > 85% and amplitude of lateral head displacement (ALH) > 2.5 µm/ s) (r = -0.711, P = 0.021) and (P < 0.05) positively correlated with sperm viability (r = 0.669, P = 0.035). Organic Zn and Cu supplementation was associated with an increase in the expression of antioxidant defense enzyme genes in bucks.

Advancement of puberty and enhancement of seminal characteristics by supplementation of trace minerals to bucks.

Attainment of puberty in animals is dependent on their age, body weight, nutritional status, genetic and environmental conditions. Nutritionally, organic minerals are suggested to improve semen production, sperm motility and male fertility. In this context, role of organic zinc (Zn) and copper (Cu) in advancing male puberty and semen characters in Osmanabadi goats were studied. Forty one (n = 41) bucks (Aged 5 months) were divided into ten groups and the dietary treatments comprised of a control group (basal diet; without additional trace mineral supplementation) and nine treatment groups that received, in addition to the basal diet, various doses of trace minerals (mg) on per kg dry matter basis, organic Zn as low Zn20, medium Zn40 and high Zn60, organic Cu as low Cu12.5, medium Cu25, high Cu37.5 and combination of organic Zn + Cu as low Zn20 + Cu12.5, medium Zn40 + Cu25, high Zn60 + Cu37.5, respectively fed for a period of 8 months. Bucks fed organic trace minerals reached puberty 28-35 days earlier than control group. In addition, improvement (P < .01) in testosterone hormone (ng/ml) levels (control: 1.63 ± 0.07 VS Zn60: 2.54 ± 0.02; Cu12.5: 6.17 ± 0.05; Cu25: 3.01 ± 0.04; Cu37.5: 2.39 ± 0.06; Zn20 + Cu12.5: 1.94 ± 0.02; Zn60 + Cu37.5: 2.44 ± 0.16 at 240 days), semen production capacity (sperm concentration, volume, mass motility) and semen quality (higher progressive motility, velocity, sperm membrane integrity and acrosome integrity) were observed in supplemented groups (P < .05) than the control bucks. The present study demonstrated that, additional feeding of organic Zn and Cu to growing male goats advanced onset of puberty and improved quantitative and qualitative semen characteristics. The results also implied that the organic Cu had a significant effect on overall performances of bucks as compared to Zn alone or Zn and Cu in combination.

Effect of suppression of plasma prolactin on luteinizing hormone concentration, intersequence pause days and egg production in domestic hen.

The aim of this study was to investigate the effects of suppression of plasma prolactin (PRL) concentration on circulating concentrations of luteinizing hormone (LH), progesterone (P(4)), estradiol (E(2)beta), pause days and egg production in birds later in the reproductive period. Twenty-four White Leghorn birds of same age group were divided into two groups of 12 in each. Birds of each group were administered s/c either with placebo (control group) or equal volumes of anti PRL agent (2-bromo-alpha-ergocriptine) solution containing at 100 microg/kg body weight/hen/week (treated group) from 72 to 82 weeks of age. Egg production and inter sequence pauses were recorded daily from both the groups. Plasma PRL, LH, E(2)beta and P(4) concentrations were estimated in blood samples collected at weekly intervals. At 77th weeks of age, blood samples from treated and control birds were obtained every 3h for 36h to study the surges of LH. It was found that plasma PRL concentration was lower (p<0.01) in bromocriptine treated birds with high concentrations of LH, its 3h LH surges, E(2)beta and P(4) in plasma. Higher egg production, less pause days in treated birds may be the result of low PRL concentration, associated with positively correlated responses of high concentrations of LH (with regular interval and duration of LH surges), E(2)beta and P(4) concentration required for completion of egg formation and oviposition. In conclusion, bromocriptine administration decreased (p<0.01) PRL concentration increased (p<0.01) steroid hormones and LH surges, for egg formation and oviposition and enabled the birds to lay more eggs even later in the productive period with the available resources under normal husbandry practices.

Effect of detoxified karanja (Pongamia spp.) cake on testicular architecture and semen production in ram lambs.

The protein-rich non-conventional detoxified karanja cake (dKC) can be used in place of conventional protein supplements like soybean meal (SBM), groundnut meal, etc. in livestock feed. The present study was conducted to assess the effect of two levels of dKC by replacing SBM on testicular architecture, semen quality and expressions of mRNAs encoding luteinizing hormone receptor (LHR) and insulin-like growth factor (IGF-I) in testes of ram lambs. Eighteen ram lambs were randomly divided into three groups (n = 6) and fed different levels (%) of karanja cake (0% replacement--control; 50% replacement--dKC-50 and 75% replacement--dKC-75) for 140 days. After 120 days of feeding, the semen from the animals was collected and analysed. The testes samples were collected on day 140 of feeding for transcripts expression studies. The dKC-50 group had no change in BW, whereas dKC-75 group showed decreased (P < 0.05) BW as compared with control. The number of animals ejaculated semen in dKC-75 group was lower (P < 0.05) than the control group. A reduction (P < 0.05) in LHR expression in dKC-75 was observed, whereas a reduction in IGF-I expression (P < 0.05) was observed in dKC-50 and dKC-75 as compared with control group. The study reveals that in ram lambs, long-term feeding of dKC at 50% replacement of SBM may not affect BW. However, long-term feeding of dKC as a replacement of SBM may affect testicular function.

Effect of prostaglandin producing modulators on in vitro growth of buffalo uterine epithelial cells.

Studies were conducted to examine the effect of seven prostaglandin producing modulators on the in vitro growth of uterine epithelial cells in buffalo. The uterine epithelial cells isolated from slaughtered buffaloes were cultured in media containing a) Lipopolysaccaride (LPS): 0, 0.01, 0.1, 1, 10 and 100 µg/ml, b) linoleic acid: 0, 0.01, 0.1, 1, 10 and 100 µg/ml, c) linolenic acid: 0, 0.01, 0.1, 1, 10 and 100 µg/ml, d) oxytocin: 0, 10, 100, 1,000, 10,000 and 100,000 nm, e) tumor necrosis factor-α (TNF-α): 0, 0.05, 0.5, 1, 2.5 and 5 nm, f) progesterone: 0.1, 10, 25, 50, 75 and 100 nM, and g) estradiol: 0, 2.5, 5, 10, 20 and 50 nM. The control medium consisted of RPMI-1640 plus 10% bovine fetal serum. The growth of uterine epithelial were measured in terms of viability, cell number increment and monolayer formation. Results suggested that the growth of uterine epithelial cells were significantly (P < 0.05) higher in media containing 10 µg/ml, 10 µg/ml, 1 nm and 10 µg/ml linoleic acid, linolenic acid, TNF-α and LPS, respectively compared to control and lower doses used. Progesterone, estradiol and oxytocin did not significantly (P > 0.05) increase the growth of uterine epithelial cells. In conclusion, the growth of uterine epithelial cells increased when exposed to modulators in the order of linoleic acid ≥ linolenic acid ≥ LPS ≥ TNF-α > progesterone > estrogen > oxytocin.

Changes in luteal cells distribution, apoptotic rate, lipid peroxidation levels and antioxidant enzyme activities in buffalo (Bubalus bubalis) corpus luteum.

Buffalo (Bubalus bubalis) is known for its weak/silent estrous behaviour, lower conception rate and longer inter-calving interval as compared to cattle. Understanding the kinetics and functional properties of luteal cells may be helpful to improve reproductive efficiency in the buffalo. Hence the present study was designed to assess the size and distribution of steroidogenic luteal cells along with biochemical properties during different phases of corpus luteum (CL) in the buffalo. The ovaries collected from the local abattoir were classified into three phases, early, mid and late, based on the morphological appearance of the CL as well as the follicles in the ovary. The proportion (%) of the luteal cells (>10microm diameter) increased (P<0.01) from early (30.7+/-1.3) to mid (36.30+/-1.6), and then decreased (P<0.01) in late luteal (31.46+/-1.8) phases. Percentage of small luteal cells (10-20microm diameter) was higher (P<0.05) in early (58.47+/-0.61) and mid (61.29+/-0.67) than late luteal (37.18+/-1.50) phases of CL. However, the percentage of large luteal cells (20-50microm diameter) was higher (P<0.05) only in late (62.82+/-1.50) than early (41.53+/-0.61) and mid (38.71+/-0.67) phases of CL. The average size (microm) of the large luteal cells increased (P<0.05) from early (25.46+/-0.62) to mid (27.15+/-0.5) and late (28.86+/-0.47) luteal phases. The percentage of luteal cells expressing in situ DNA fragmentation was significantly (P<0.05) higher in the late luteal (41.17+/-5.8) than mid-luteal (21.15+/-4.9) phase of the CL. In the early stage, half of the steroidogenic luteal cells had significantly (P<0.05) less 3beta-HSD activity than the other two phases. In the mid stage, the steroidogenic luteal cells had significantly higher (P<0.05) intense 3beta-HSD activity than the other two phases. Further in the late phase, a significant (P<0.05) reduction in intense 3beta-HSD activity was observed in the large luteal cells. The lipid peroxidation (micromol/g of CL) levels were significantly (P<0.05) higher in late luteal (3.46+/-0.2) than the mid-luteal (1.43+/-0.16) phases. The superoxide dismutase and catalase enzyme levels (U/mg of protein) were also significantly (P<0.05) higher in late luteal (0.9+/-0.015 and 3.37+/-0.45, respectively) than the mid-luteal (0.1+/-0.01 and 2.34+/-0.3, respectively) phases. In contrast, the GPx activity (U/mg of protein) decreased significantly (P<0.05) from mid-luteal (1.85+/-0.4) to late luteal (1.22+/-0.2) phases. The present study suggests that (i) the decrease in progesterone levels in late CL may be associated with loss of 3beta-HSD activity in large luteal cells and (ii) demise of the buffalo CL may be mediated by apoptosis despite the high levels of luteal antioxidant enzymes.

Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro.

The objective of the present experiment was to examine the influence of mean physiological concentration of insulin-like growth factor-I (IGF-I) on frozen-thawed Surti buffalo (Bubalus bubalis) spermatozoa functional parameters, i.e., motility, plasmalemma integrity, acrosomal integrity, functional membrane integrity, lipid peroxidation and fructose uptake in vitro. Frozen-thawed semen samples (n=6) were washed in tris buffer and divided into two equal parts (control and IGF-I groups). Only in the IGF-I group, IGF-I (rhIGF-I analogue) was added to a final concentration of 100 ng/ml. The samples were incubated at 37 degrees C for 2h and the assessments were made at 0, 30, 60, 90 and 120 min of incubation. The mean concentration of the buffalo seminal plasma (n=17) IGF-I was 116.83+/-28.34 ng/ml (range 41.4-198.95). IGF-I had significant effect on the total motility (P<0.01), progressive forward motility (P<0.01), functional membrane integrity (P<0.05) and lipid peroxidation levels (P<0.05) during the 120-min study period as assessed by area under curve. Treatment with IGF-I increased (P<0.01) the total spermatozoa motility at 30, 60 and 90 min as compared to the control. The progressive forward motility was significantly (P<0.01) higher at 60 and 90 min of incubation. The addition of IGF-I resulted in significant (P<0.01) increase in straight-line velocity (VSL, microm/s) as compared to the control at 60 and 90 min of incubation. The linearity (%) was significantly (P<0.01) higher in IGF-I treated semen as compared to control at 60 min of incubation. Plasmalemma integrity in IGF-I group was significantly (P<0.05) higher than control at 30 and 60 min of incubation. The functional membrane integrity differed significantly (P<0.01) between groups (control and IGF-I) at 60 and 90 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant (P<0.05) loss of acrosome was observed at 60 and 90 min of incubation in control (63.87+/-3.17 vs. 58.52+/-2.54) and IGF-I (61.60+/-2.26 vs. 56.11+/-2.12) groups, respectively. Lipid peroxidation levels were significantly lower in IGF-I group at 90 min (P<0.05) and 120 min (P<0.01) of incubation than the control group. Fructose utilization was significantly higher in IGF-I group as compared to control at 30 min (P<0.05) and 60 min (P<0.01) of incubation. The present study suggests that addition of IGF-I improve spermatozoa functional parameters by reducing lipid peroxidation levels.

The possible role of prolactin in laying performance and steroid hormone secretion in domestic hen (Gallus domesticus).

The aim of this study was to investigate the basic physiological mechanism involved in taking pauses between the sequences of egg laying in domestic hen to improve egg production by extending the sequence length and decreasing the intersequence pause days by modulating the prolactin concentration in birds. Fifty healthy female white leghorn birds were administered anti-prolactin agent (2-bromo-alpha-ergocriptine, Sigma, USA) subcutaneously at 100 microg/kg body weight at weekly intervals from 17th to 36th week of age. Another group of fifty birds was given placebo in place of the modulating agent. The level of prolactin remained lower in the treated birds than in the control birds throughout the production cycle up to 72 weeks of age. The level of prolactin in the control group was found to decrease during the peak production period. The average percentage of egg production from 19 to 72 week period was 87.67 in the treatment group as compared to 83.56 in the control group. Oestradiol-17beta and progesterone concentrations in the treated birds were significantly (P<0.01) higher than those in control birds, during and after withdrawal of the treatment. Prolactin level was negatively correlated with egg production (r=-0.02; r=-0.12) and with oestradiol-17beta (r=-0.75; r=-0.38) and progesterone (r=-0.20; r=-0.83), respectively, in control and treatment groups. The total number of pause days during the production period decreased significantly (P<0.01) in the treatment group, resulting in a 4.11% increase in egg production. It is concluded that there is a consistent relationship between plasma prolactin in the physiological range and laying performance in domestic hen.