Effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred ram in semi-arid tropics.

A prolific three-breed (Malpura, Patanwadi, and Garole) cross Avishaan sheep has been developed in the semi-arid zone to improve farmer's income. Nutritional scarcity is a major limitation in animal husbandry during the dearth period of semi-arid tropics. Therefore, before the inaugural launch of the breed into the field, a study was designed to evaluate the effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred rams in semi-arid tropics. Thus, 16 native adapted (Malpura) and 16 native-crossbred rams were equally distributed into four groups, namely, native control (G1), native nutritional stress (G2), native-crossbred control (G3), and native-crossbred nutritional stress (G4). Both the control groups (G1 and G3) were kept on their maintenance requirement as per their body weight, whereas the nutritional stress groups (G2 and G4) were provided 30% less than their maintenance requirement. The body weight of G4 decline (P<0.05) as compared to their initial weight. The plasma glucose level of G2 and G4 reduced (P<0.05) in comparison with G1 and G3, respectively. The total motile sperm percentage, rapid motile sperm percentage, and sperm viability decrease significantly (P<0.05) within the acceptable limit in native-crossbred rams (G4) under nutritional scarcity. However, the similar blood biochemical along with acceptable seminal attributes of all the rams reflected that native-crossbred rams can cope with the nutritional scarcity in semi-arid tropics and have the potential to contribute to the sustainable small ruminant production system for livelihood security in this region.

Combined effect of heat and nutritional stress on superovulation of Malpura ewes in a semi-arid region.

Sheep reared in hot semi-arid environments are generally exposed to heat and nutritional stress in some seasons of the year, which affects both production and reproduction. To assess the effect of high ambient temperature and feed scarcity on superovulation, 16 adult Malpura ewes were randomly divided into two groups of 8 animals each. G1 (control) was kept under a shed and offered a maintenance diet, and G2 (combined stress) was subjected to both nutritional (30% less of maintenance diet) and heat (38-44 °C for 6 h/day) stress. Ewes were superovulated without estrus synchronization by a combination of single injection of 200 IU eCG and 8 injections of FSH (Folltropin-V) at 12-h intervals in tapering doses of 5 mg/kg body weight, starting from the day 7 of natural estrus. eCG was given with the first injection and PGF2α (10 mg) was given with the second last FSH injection. G2 increased respiration rate and rectal temperature (P < 0.01), and blood urea level (P < 0.05), whereas it decreased average daily gain, plasma T4 concentration (P < 0.01) and body weight (P < 0.05). Plasma estradiol level was lower (P < 0.05) in G2 ewes as compared to control (G1) ewes. However, the number of ewes showed a superovulatory response (88 vs 66% ewes ≥ 3 corpus luteum), ovulation rate (8.75 vs 5.88) and embryo production (5.5 vs 3.9) decreased, and the number of large follicles (anovulation) increased (1.0 vs 2.14) in G2 ewes. G2 had a comparable effect on the superovulatory response compared to control ewes although physiological changes occurred as an adaptive mechanism to stress. Therefore, the well-adapted cyclic sheep of the semi-arid region may be used for superovulation despite the stressful condition of heat exposure and nutritional insufficiency.

Molecular characterization of GPR50 gene and study of its comparative genetic variability in sheep breeds adapted to different thermo-contrasting climatic regimens.

GPR50, formerly known as a melatonin-related receptor, is one of the three subtypes of melatonin receptor subfamily, together with MTNR1A and MTNR1B. GPR50, despite its high identity with the melatonin receptor family, does not bind melatonin and is considered to be an ortholog of MTNR1C in mammals. GPR50-expressing cells have been found in the dorsomedial nucleus of the hypothalamus, the periventricular nucleus, and the median eminence. Genetic and functional evidence have been recently investigated linking GPR50 to adaptive thermogenesis and torpor, but still, it is an orphan receptor and is yet to be studied conclusively. The aims of the study were to characterize the GPR50 gene of sheep and to study the sequence variability of the gene in Indian sheep breeds of two different thermo-varied agroclimatic conditions. Genomic DNA isolation was done and a 791-bp sequence was amplified using self-designed primers and SNP profiling done out of samples of all the breeds to study the relative frequency of SNPs in each of the breed. Five important non-synonymous mutations were observed in the various breeds studied. T698G, G1097A, G1270A, G1318A, and C1334G lead to the following substitution: valine by glycine, arginine by glutamine, threonine by alanine, isoleucine by valine, and serine by cytosine, respectively. Two synonymous mutations (T663G and C888T) were also observed in some of the studied breeds. G1270A and C888T were the most prevalent SNPs observed in nearly all of the breeds. C888T SNPs were observed in higher prevalence in Chokla, Marwari, and Magra in comparison to Gaddi and Bharat Merino. A PolyPhen-2 analysis, which is used to assess the potential damaging nature of an SNP, revealed that mutation T698G and G1270A were benign while G1097A, G1318A, and C1334G were damaging with a score of 0.987, 0.993, and 0.739, respectively. A 3-D homology model of the protein was prepared using c4zwjA (UniProt sequence ID) as a template using the online version of Phyre2 protein modeling software. The structure demonstrated closed similarity with other G-coupled receptor and it had a 45 % α-helical content. G1270A and C888T may be taken up for SNP correlation in a larger population study for their association with heat stress protection.

Impact of heat stress, nutritional restriction and combined stresses (heat and nutritional) on growth and reproductive performance of Malpura rams under semi-arid tropical environment.

A study was conducted to assess the combined effect of heat stress and nutritional restriction on growth and reproductive performances in Malpura rams. Twenty-eight adult Malpura rams (average body weight (BW) 66.0 kg) were used in this study. The rams were divided into four groups: CON (n = 7; control), HES (n = 7; heat stress), NUS (n = 7; nutritional stress) and COS (n = 7; combined stress). The study was conducted for a period of 2 months. CON and HES rams had ad libitum access to their feed while NUS and COS rams were under restricted feed (30% intake of CON rams) to induce nutritional stress. The HES and COS rams were kept in climatic chamber at 42 °C and 55% relative humidity for 6 h a day between 10 : 00 h and 16 : 00 h to induce heat stress. Body weight increased significantly (p < 0.05) in CON as compared to NUS and COS. When compared within groups, scrotal width morning, scrotal width afternoon, scrotal circumference morning and scrotal circumference afternoon were significantly (p < 0.05) larger in CON while smaller in COS rams. The higher testicular length was recorded both during morning (p < 0.05) and afternoon (p < 0.01) in COS rams while the lowest in NUS rams. The highest plasma testosterone concentration was recorded in CON and lowest in COS rams. Semen volume and mass motility also differed significantly (p < 0.05) between the groups. The highest semen volume and mass motility was recorded in CON and NUS while lowest in both HES and COS rams. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on reproductive performance of rams.

Study of conformational properties of solid phase synthesized ovine kisspeptin-14 using Circular Dichroism spectroscopy.

Kisspeptins are peptide products of Kiss-1 gene and have been substantially associated with the initiation of puberty by virtue of their ability to cause pulsatile release of GnRH. Kisspeptin consists of 54 amino acids domain called metastin and its biological activity may be localized to the C-terminal (C-10, C-13, and C-14) segments. Kisspeptin binds to its cognate receptor GPR54 in the hypothalamic neurons and it is a G-coupled membrane receptor. This study is an attempt to understand the tentative conformation of the peptides in its native membrane mimicking environment. A 14 amino-acid derivative of kisspeptin (Asp-58-Val59-Ser60-Ala61-Tyr62-Asn63-Trp64-Asn65-Ser66-Phe67-Gly68-Leu69-Arg70-Tyr71NH2) was synthesized by f-moc (9-fluorenyl methoxy carbonyl) solid phase synthesis strategy. The synthetic peptide was cleaved and purified by Reverse phase-HPLC. CD spectroscopic analysis of secondary structure of the peptide revealed random coil disordered conformation in the aqueous environment. However, the peptide adopted more ordered ß-sheet conformation in the solvents such as TFE and HFIP.

Characterization of MTNR1A gene in terms of genetic variability in a panel of subtemperate and subtropical Indian sheep breeds.

Seasonality of animals is an important adaptive trait for successful survival and production during limited food availability and extreme environmental conditions. Photoperiodic changes in day length are utilized by these seasonal animals as an important environmental cue for regulating their annual rhythms of reproduction cycles. Melatonin is an important hormone which is secreted by the pineal gland in proportion to darkness and its effect is mediated by melatonin receptor subtypes, principally MTNR1A. In the present study, polymorphism in the coding sequence at two important SNPs (C606T and G612A),known to be markers for out of season breeding in sheep were studied by PCR-RFLP in a panel of four breeds of sheep from subtemperate and subtropical arid conditions, respectively. The frequencies of 'G' and 'A' alleles with reference to G612A SNP did not differ considerably among all the breeds of sheep. Frequency of 'T' allele of the C606T SNP was found to be dom inantly higher in subtemperate sheep breeds in comparison to subtropical sheep breeds. Identified SNPs in the coding region were mostly synonymous and did not lead to any change in conformation of the MTNR1A receptor protein.

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Development of PCR-based technique for detection of purity of Pashmina fiber from textile materials.

Pashmina fiber is one of major specialty animal fiber in India. The quality of Pashmina obtained from Changthangi and Chegu goats in India is very good. Due to restricted availability and high prices, adulteration of natural prized fibers is becoming a common practice by the manufacturers. Sheep wool is a cheap substitute, which is usually used for adulteration and false declaration of Pashmina-based products. Presently, there is lack of cost-effective and readily available methodology to identify the adulteration of Pashmina products from other similar looking substitutes like sheep wool. Polymerase chain reaction (PCR)-based detection method can be used to identify origin of animal fiber. Extraction of quality DNA from dyed and processed animal fiber and textile materials is a limiting factor in the development of such detection methods. In the present study, quality DNA was extracted from textile materials, and PCR-based technique using mitochondrial gene (12S rRNA) specific primers was developed for detection of the Pashmina in textile blends. This technique has been used for detection of the adulteration of the Pashmina products with sheep wool. The technique can detect adulteration level up to 10 % of sheep/goat fibers in textile blends.

Effect of milk replacer and rumen inert fat on growth and reproduction of Malpura ram lambs.

The objective of this study was to assess the effects of milk replacer and rumen inert fat on growth, testicular development, puberty, semen production and sperm motion characteristics of ram lambs reared under intensive management in semi-arid climatic conditions. Seven-day-old male lambs of Malpura breed (n=20) were divided equally into two groups. Up to weaning, the lambs in G1 group (control) were fed concentrate, green khejri (Prosopis cineraria) leaves and cowpea (Vigna unguiculata) hay along with suckling of dams, whereas lambs in G2 group were fed reconstituted milk at 17 g/lamb per day for the 1st week and at 34 g/lamb per day from 2nd week in addition to the feed inputs given in G1. During post weaning, lambs in the G1 group were given control concentrate, whereas in G2 the control concentrate supplemented with 40 g rumen inert fat per kg of feed was offered along with dry pala (Zizyphus nummularia) and ardu (Ailanthus excelsa) leaves. BWs of lambs were recorded weekly up to 6 months of age. Ram lambs of both the groups were trained for semen collection at a weekly interval from the age of 5 months and simultaneously testicular measurements were recorded fortnightly. The feeding of milk replacer and rumen inert fat had positive (P<0.05) effects on BW, testicular length, testicular volume, semen volume, sperm concentration, mass motility, % motility, % rapid, medium or slow motile spermatozoa. However, no significant effect was observed on testicular breadth, scrotal circumference, age of puberty, sperm velocities and other CASA-derived parameters. The results of this study indicate that higher plane of nutrition in the form of milk-replacer feeding during preweaning and rumen inert fat-supplemented feed during the postweaning period to growing ram lambs enhances their growth, testicular development and semen quality.

Effect of mineral mixture and antioxidant supplementation on growth, reproductive performance and adaptive capability of Malpura ewes subjected to heat stress.

This study was conducted to evaluate the effect of mineral and antioxidant supplementation on growth, reproductive performance and physiological adaptability of heat-stressed Malpura ewes. The study was conducted for a period of 21 days in 21 adult Malpura ewes. The ewes were randomly divided into three groups with seven animals each viz. GI (control; n = 7), GII (heat stress; n = 7) and GIII (heat stress + mineral and antioxidant supplementation; n = 7). The animals were stall fed ad libitum with the diet consisting of 70% roughage and 30% concentrate. GI ewes were maintained under normal controlled condition in the shed, while GII and GIII ewes were subjected to heat stress by exposing them to 42 °C in the climatic chamber. The parameters studied were feed intake (FI), water intake (WI), body weight, body condition score (BCS), physiological, biochemical and endocrine responses. Heat stress significantly altered FI, water intake, BCS, respiration rate and rectal temperature in the afternoon, oestrus duration, estradiol, progesterone, Hb, PCV, plasma glucose, total protein, cortisol, T3 and T4 levels while mineral and antioxidant supplementation ameliorated this heat stress effect on the parameters studied. Further, the adverse effect of heat stress on the productive and reproductive efficiency of Malpura ewes was reduced considerably by mineral mixture and antioxidant supplementation. This is evident from the non-significant difference in BCS, oestrus duration and plasma estradiol between GI and GIII in this study. Hence, it is very pertinent to conclude from this study that mineral mixture and antioxidant supplementation were able to protect Malpura ewes against heat stress.

Sequence analysis and identification of new variations in the coding sequence of melatonin receptor gene (MTNR1A) of Indian Chokla sheep breed.

Melatonin receptor 1A gene is the prime receptor mediating the effect of melatonin at the neuroendocrine level for control of seasonal reproduction in sheep. The aims of this study were to examine the polymorphism pattern of coding sequence of MTNR1A gene in Chokla sheep, a breed of Indian arid tract and to identify new variations in relation to its aseasonal status. Genomic DNAs of 101 Chokla sheep were collected and an 824 bp coding sequence of Exon II was amplified. RFLP was performed with enzyme RsaI and MnlI to assess the presence of polymorphism at position C606T and G612A, respectively. Genotyping revealed significantly higher frequency of M and R alleles than m and r alleles. RR and MM were found to be dominantly present in the group of studied population. Cloning and sequencing of Exon II followed by mutation/polymorphism analysis revealed ten mutations of which three were non-synonymous mutations (G706A, C893A, G931C). G706A leads to substitution of valine by isoleucine Val125I (U14109) in the fifth transmembrane domain. C893A leads to substitution of alanine by aspartic acid in the third extracellular loop. G931C mutation brings about substitution of amino acid alanine by proline in the seventh transmembrane helix, can affect the conformational stability of the molecule. Polyphen-2 analysis revealed that the polymorphism at position 931 is potentially damaging while the mutations at positions 706 and 893 were benign. It is concluded that G931C mutation of MTNR 1A gene, may explain, in part, the importance of melatonin structure integrity in influencing seasonality in sheep.

Effect of multiple stresses (thermal, nutritional, and walking stress) on the reproductive performance of malpura ewes.

A study was conducted to assess the effect of multiple stresses (thermal, nutritional, and walking stress) on the reproductive performance of Malpura ewes. The study was conducted for a period of 35 days covering two estrous cycles during summer season (April-May). The ewes were randomly allocated into two groups of twelve animals each namely, GI (n=12; Control), and GII (n=1 2; Multiple stresses). GI ewes were maintained in the shed, while GII ewes were subjected to multiple stresses (thermal, nutritional, and walking stress). The estrus % differed significantly (P< 0.05) between the groups. Estrus duration also showed similar trend to that of estrus %. Both conception and lambing rate also differed significantly (P< 0.05) between the control and multiple-stress group. In addition plasma estradiol and progesterone also showed significant difference between the groups. The study proved the detrimental effects of multiple-stresses on various reproductive parameters studied. Hence it is very pertinent to conclude that when two or more than two stressors occur simultaneously, the total impact may be severe on reproductive functions of the animals.

Effect of concentrate supplementation on performance, ovarian response, and some biochemical profile of Malpura ewes.

Effect of feed flushing on ovulation rate was investigated during the autumn seasons on 24 adult Malpura ewes (BW 34.8 ± 0.58 kg and age 4-7 years) equally divided into two groups. Ewes in G1 (group 1) were grazed 8-10 h daily on Cenchrus ciliaris pasture interspersed with seasonal shrub. In addition to grazing, concentrate was provided at 1.5% of BW to the animals in G2 (group 2) for a period of 35 days. Estrus was synchronized by a double injection schedule of PGF(2α) at 0 and 10 days of the experiment and detected by parading aproned rams at 6 h intervals. Blood samples were collected at weekly interval during the estrous cycle. A rumen fermentation study was conducted on day 23 of the experiment at 0 and 4 h post-concentrate feeding. Ovarian responses in terms of number of corpora lutea and large follicles was examined on all the ewes by laparoscopy after 3 to 6 days of each estrus and were found to be similar in both the groups. Hemoglobin and packed cell volume, total protein, albumin, and globulin were similar among the groups. Concentration of plasma glucose (12 and 22 days) was higher (P < 0.05) in G2 vis-à-vis control. Plasma urea was higher (P < 0.01) in the control than G2. Rumen liquor pH, concentration of total N, TCA-ppt N, NH(3)-N, and TVFA were significantly higher (P < 0.01) in G2 than the control. Thus, it can be concluded that concentrate supplementation in ewes prior to mating (flushing) did not enhance ovulation response during the autumn season.

Effect of thermal stress, restricted feeding and combined stresses (thermal stress and restricted feeding) on growth and plasma reproductive hormone levels of Malpura ewes under semi-arid tropical environment.

A study was conducted to assess the effect of thermal, nutritional and combined stresses (thermal and nutritional) on the growth, oestradiol and progesterone levels during oestrus cycles in Malpura ewes. Twenty-eight adult Malpura ewes were used in the present study. The ewes were randomly allocated into four groups, viz., GI (n=7; control), GII (n=7; thermal stress), GIII (n=7; restricted feeding) and GIV (n=7; combined stress). The animals were stall fed with a diet consisting of 60% roughage and 40% concentrate. GI and GII ewes were provided with ad libitum feeding while GIII and GIV ewes were provided with restricted feed (30% intake of GI and GII ewes) to induce nutritional insufficiency. GII and GIV ewes were kept in climatic chamber at 40°C and 55% RH for 6 h a day between 10:00 and 16:00 hours to induce thermal stress for a period of two oestrous cycles. Parameters studied were body weight, oestrus incidences, plasma oestradiol 17-ß, plasma progesterone, conception rate, gestation period, lambing rate, and birth weight of lambs. The results indicate that combined stress significantly (p<0.05) reduced body weight, oestrus duration, birth weight of lambs, and oestradiol 17-ß whereas significantly (p < 0.05) increased oestrus cycle length and progesterone. Furthermore, the results reveal that on comparative basis, ewes were able to better adapt in terms of growth and reproduction to restricted feeding than thermal stress. However, when restricted feeding was coupled with thermal stress it had significant (p<0.05) influence on body weight, average daily gain, oestradiol 17-ß and progesterone concentrations. This showed that combined stress were more detrimental for these reproductive hormones in Malpura ewes under a hot semi-arid environment.

Effect of induced body condition score differences on sexual behavior, scrotal measurements, semen attributes and endocrine responses in Malpura rams under hot semi-arid environment.

The study was conducted for a period of 45 days to assess the influence of body condition scoring (BCS) on reproductive efficiency of Malpura rams in terms of sexual behaviour, scrotal measurements, semen attributes and endocrine responses. The rams were randomly divided into three groups as group I (BCS = 2.5; n = 7), group II (BCS = 3.0; n = 7) and group III (BCS = 4.0; n = 7). Apart from grazing for 8 h a day, the rams were provided with concentrate mixture at the rate of 100, 300 and 500 g to groups I, II and III respectively to attain their respective BCS. Under these differential feeding regimes, sexual behaviour scrotal measurements, semen characteristics and endocrine parameters were studied at 15-days interval. Sexual behaviour, scrotal volume, scrotal circumference, testicular width, testicular length and scrotal skin thickness differed significantly (p < 0.05) between lower BCS group (group I) and higher BCS groups (groups II and III). The semen volume, mass motility and progressive sperm motility were recorded significantly (p < 0.05) higher in group II and lowest in group I. However, the sperm concentration did not differ significantly between the groups. The mean plasma testosterone concentration differed significantly (p < 0.05) between lower BCS group (group I) and higher BCS groups (groups II and III). Circulating plasma hormone concentrations were influenced due to the BCS. Plasma cortisol, T(3) and T(4) increased as the BCS improved from lower to higher. In conclusion, better reproductive efficiency of rams of moderate BCS (3.0 and 3.5) than rams of lower (2.5) and higher BCS (4.0) indicates the importance of maintaining optimum body condition of breeding rams under hot semi-arid environment.

Objective assessment of sperm motion characteristics of Malpura ram lambs raised under intensive management system in semiarid tropical environment.

A study was conducted to evaluate the semen production and sperm motion characteristics of ram lambs by computer-aided semen analysis technique. Eight Malpura rams were raised under intensive management system and were trained for semen collection at a weekly interval from the age of 6 months. Rams were scheduled for semen collection at a weekly interval up to 1 year of age to assess their potential for semen production and objective evaluation of semen quality. The average age of ram lambs at the time of first ejaculation was 219 days ranging from 186 to 245 days. The age of ram lambs significantly (p < 0.05) influenced sperm concentration, sperm velocities, and beat frequency of spermatozoa, which were higher in 9-12-month-old compared to 6-9-month-old ram lambs. However, the effect of age was not significant on semen volume, percent motility, percent rapid, medium or slow motile spermatozoa, percent linearity, percent straightness, amplitude of lateral head displacement, percent elongation, and area of sperm head. The body weight of ram lambs was significantly (p < 0.01) and positively correlated (r = 0.46) with age. The results indicate that Malpura ram lambs of 9-12 months of age raised under the intensive management system in a semiarid tropical environment can produce good quality of semen.

Effect of induced body condition score differences on physiological response, productive and reproductive performance of Malpura ewes kept in a hot, semi-arid environment.

This study was undertaken to study the influence of induced body condition score (BCS) differences on physiological response, productive and reproductive performance of Malpura ewes to optimise BCS for these ewes for maximising production making it economically viable. The study was conducted for a period of 1 year using thirty healthy Malpura ewes (2-4 year old). The animals were randomly divided and different BCS was induced within three groups named Group I (BCS 2.5; n = 10), Group II (BCS 3.0-3.5; n = 10) and Group III (BCS 4.0; n = 10). The parameters included in the study were allometric measurements, physiological response, wool yield and reproductive performance. BCS had a significant influence on allometric measurements, respiration rate and different reproductive parameters studied, while wool production differed significantly during spring and non-significantly during autumn. The results revealed that the reproductive performance of Malpura ewes with a BCS of 3.0-3.5 was better in comparison with the groups with lower and higher BCS. It may be concluded from this study that an active management of breeding sheep flock to achieve a BCS of 3.0-3.5 may prove to result in an economically viable return from these flocks.

Effect of controlled and uncontrolled rate of cooling, prior to controlled rate of freezing, on motion characteristics and acrosomal integrity of cryopreserved ram spermatozoa.

A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.

Sperm motion characteristics of GarolexMalpura sheep evolved in a semi-arid tropical environment through introgression of FecB gene.

The arid and semi-arid tropical climates of India are endowed with vast diversity of non-prolific sheep breeds. The GarolexMalpura sheep has been evolved in a semi-arid tropical environment by introgression of FecB gene via artificial insemination of Malpura ewes using diluted semen of prolific microsheep Garole and subsequently multiplied by inter se mating among GarolexMalpura halfbreds. The aim of the present study was to identify FecB mutation in sexually mature GarolexMalpura rams by forced RFLP-PCR of BMPR-1B gene and evaluate: (i) semen production and sperm motion characteristics of GM rams and (ii) influence of age and FecB genotype on their semen attributes. Semen was collected during autumn season from 12 donor rams by artificial vagina on 8 occasions at weekly interval. The overall means of traits which did not differed significantly with age or FecB genotyping were volume (0.72 ml), mass motility (4.44), sperm concentration (2721.56 x 10(6)ml(-1)), curvilinear velocity (134.51 microm/s), motility (81.3%), amplitude of lateral head displacement (6.24 microm), beat frequency (44.43 Hz), sperm head elongation (48.9%) and sperm head area (10.01 microm(2)). The FecB genotyping had a significant effect (P<0.05) on percent linearity and rapid motile sperms, which did not vary significantly with age. Although sperm concentration was higher in FecB(BB) and FecB(B+), compared to FecB(++) genotypes but the effect was non-significant. The age and FecB genotyping had significant effects (P<0.05) on straightness, average path velocity, straight-line velocity and percentage of medium or slow motile sperms. It is concluded that GarolexMalpura rams with introgressed FecB gene are capable of producing good quality semen in a semi-arid tropical climate.

Production of prolific microsheep by embryo transfer into large non-prolific sheep.

The Garole is a prolific breed of microsheep that possesses the FecB gene, which increases ovulation rate. The purpose of this study was to compare embryo production by multiple ovulation in seven Garole ewes with that in seven normal size, non-prolific Malpura ewes, and assess the influence of the large body size of Awassi crossbred recipient ewes on the birth-weight of Garole lambs. Oestrus was synchronised with two intramuscular injections of 7.5 mg prostaglandin F(2alpha) administered 10 days apart. The donor ewes were superovulated by the use of pregnant mare serum gonadotrophin and follicle-stimulating hormone. The onset and duration of oestrus were similar in both breeds. The Garole donors had higher total mean (se) ovarian responses (15.6 [3.6] v 9.1 [2.3]), ovulation rate (13.6 [3.1] v 8.4 [2.2]) and produced more transferable embryos (6.0 [3.5] v 4.0 [0.9]) than the Malpura donors, but the differences were not statistically significant. The Garole lambs produced by embryo transfer were on average 57.8 per cent heavier at birth than contemporary Garole lambs produced by natural mating.