Rumen microbial variation and nutrient utilisation in mithun (Bos frontalis) under different feeding regimes

The aim of the study was to investigate the effect of feeding different diets on fermentation, enzyme activities and microbial population in the rumen fluid of mithun (Bos frontalis). In a randomized block design, 20 male mithun (6–8 months of age, 152 ± 12.6 kg body weight) were randomly divided into four experimental groups (n = 5/group) and fed experimental diets ad libitum for 180 days. The diet R₁ contained tree foliages (TF), R₂ comprised of 50% concentrate mixture (CM) and 50% TF, R₃ contained 50% CM and 50% rice straw, and R₄ contained 50% CM, 25% TF and 25% rice straw. Rumen liquor was collected at 0 and 180 days of the experiment for estimation of different ruminal parameters and a digestion trial was conducted at the end of the experiment. Rumen fluid was analysed for pH, ammonia nitrogen (NH₃‐N), total‐N, ruminal enzymes, short chain fatty acid (SCFA) and microbial profile. The relative quantification of ruminal microbes was carried out with real‐time PCR using bacteria as the house keeping gene. The dry matter intake, nutrients digestibility, body weight gain, NH₃‐N, total‐N, carboxymethyl cellulase, avicelase, xylanase, amylase, protease and molar proportion of butyrate were (p < 0.05) higher in mithun fed R₂, R₃ and R₄ compared to those fed R₁ diet. In contrast, increased (p < 0.05) ruminal pH, molar proportion of acetate and, acetate to propionate ratio was recorded in mithun fed only TF than those fed concentrate supplemented diets. Similarly, an increase (p < 0.05) in the population of Fibrobacter succinogenes, Ruminococcus flavefaciens and total bacteria were evident in mithun fed R₂, R₃ and R₄ compared to those fed R₁. Therefore, it is concluded that TF 25% and/or rice straw 25% along with CM 50% may be fed to the growing mithun for improved rumen ecology, nutrient utilization and thus better performance under stall fed system.     

Milk protein polymorphisms in cattle (Bos indicus), mithun (Bos frontalis) and yak (Bos grunniens) breeds and their hybrids indigenous to Bhutan

In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β‐lactoglobulin (A, B, E and M), five molecular types of α_S1‐casein (A, B, C, E and X) and three molecular types of k‐casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24–40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle.     

Plasma growth hormone concentrations in female mithun (Bos frontalis) of different ages: relations with age and body weight

The role of growth hormone (GH) in postnatal somatic growth is well established. Its basal level and relation to growth performance in different age group mithun (Bos frontalis), a semiwild ruminant has not been characterized until now. To estimate the normal blood GH level and also to assess the influence of age and body weight (BW) on blood GH level in captive mithuns, a total of 65 female mithuns was divided into six age groups (group I, 0-6 months; group II, >6-12 months; group III, >1-2 years; group IV, >2-2.5 years; group V, >2.5-3.0 years and group VI, >3.0 years). Blood samples collected weekly for six consecutive weeks were assayed for GH. GH was also estimated in the samples collected from six growing mithuns at -60, -45, -30, -15, -10, -5 and 0 min prior to GH-releasing hormone (GHRH) administration for calculation of basal GH level and at 5, 10, 15, 30 min and thereafter at 15-min interval up to 8 h post-GHRH to assess blood GH response following GHRH administration in growing mithuns. For calculation of basal plasma GH in adult mithuns, GH was measured in blood samples collected at 30-min interval for 24 h from four animals. BW of all animals was recorded on two consecutive days per week and average of weekly BW was considered for growth rate calculation. It was found that both mean GH and GH per 100 kg BW between the age groups differ (p < 0.01). With increasing age and BW, GH and GH per 100 kg BW both decreased (p < 0.01). The age group with higher plasma GH and GH per 100 kg BW showed higher growth rates (r = 0.83 and 0.97 respectively). Interestingly, mean plasma GH for six consecutive weeks in all the groups showed much greater GH concentration (group I, 86.6 +/- 9.7 ng/ml to group VI 33.2 +/- 5 ng/ml) than reported in other species. Mean basal plasma GH calculated in growing and adult mithuns was 29.6 +/- 4.01 ng/ml and around 25 +/- 3.6 ng/ml respectively. The GH peak (444 +/- 21.3 ng/ml) was registered at 15 min post-GHRH administration in growing mithuns. In conclusion, age and BW influence plasma GH and GH per 100 kg BW but the latter is a better indicator of growth. The basal plasma GH and GH response to GHRH administration is six to eight and four to five times higher in mithun than in other species reported so far. An accurate assessment of the relationship between GH profiles and protein metabolism, proper receptor level study for GH action at the cellular level and the interaction of GH with other growth factors awaits better understanding of higher GH in this unique species.     

Effect of feeding Lagerstroemia speciosa and conventional fodder based rations on nutrient utilization,ruminal metabolites and body weight gain in mithun (Bos frontalis)

The aim of the present study was to evaluate the effect of feeding green fodder, rice straw and concentrate‐based total mixed rations (TMR) on dry matter (DM) intake (DMI), nutrient utilization, rumen fermentation patterns and body weight (BW) gain (BWG) in mithun (Bos frontalis) calves. In a randomized block design, male mithun calves (n = 18, 8–10 months of age, 121 ± 2 kg BW) were randomly divided into three experimental equal groups (six animals in each group) and fed isonitrogenous TMRs ad libitum for 120 days. The TMR₁ contained 30% Napier grass and 30% rice straw, TMR₂ contained 60% rice straw and TMR₃ contained 30% tree leaves (Lagerstroemia speciosa) and 30% rice straw (DM basis). All the TMRs contained 40% concentrate mixture (DM basis). The results indicated that the BWG, DMI and feed conversion efficiency were significantly (p < 0.01) increased with the inclusion of green fodder in TMRs. The apparent digestibility of DM, crude protein, ether extract, crude fibre and nitrogen free extract were also improved significantly (p < 0.01) with the inclusion of green fodder in TMRs. The higher concentration of total nitrogen and total volatile fatty acid in rumen liquor, but low ruminal pH were evident in animals fed green fodder supplemented TMRs. An increased (p < 0.01) molar proportion of acetic acid was evident in animals fed rice straw‐based TMR. In contrast, the molar proportion of propionic and butyric acids were significantly (p < 0.01) higher in animals fed green fodder supplemented TMRs. On the basis of higher DMI and higher daily BWG, it is concluded that Napier grass and L. speciosa tree leaves may be incorporated upto 30% (DM basis) in TMR of growing mithuns for feeding in complete confinement system.     

Effects of low‐density lipoproteins as additive on quality parameters and oxidative stress following cryopreservation of mithun (Bos frontalis) spermatozoa

Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p < .05) improvement in viability, sperm and nuclear abnormality, acrosome integrity, motility (progressive and in cervical mucus), cholesterol content, and reduction in the leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes was protected significantly (p < .05) in addition to significant (p < .05) improvement in binding per cent and binding index in IVF assay in extender containing 8% LDL. These results demonstrate that although cryopreservation of mithun's spermatozoa in EY was comparable with other species, addition of 8% LDL holds a clear advantage over EY or 10% LDL.     

Effect of method and time of first colostrum feeding on serum immunoglobulin concentration, health status and body weight gain in mithun (Bos frontalis) calves

The effect of method and time of first colostrum feeding on the concentration of serum immunoglobulin (Ig) was evaluated in mithun (Bos frontalis) calves. The hypotheses were that the variable method and time of first colostrum feeding might affect the level of serum Ig and in turn the growth performance and health status of the claves during the early age. The newborn calves were randomly allotted to one of the four experimental groups - G-1: allowed to suckle the dam at own choice, G-2: separated immediately after birth and allowed to suckle the dam first at 6 h and then at own choice, G-3: bottle fed ad libitum colostrum of its own dam first at 6 h and then at 6-h intervals until 24 h, G-4: bottle fed ad libitum colostrum of its own dam within 1 h, at 6 h and then at 6-h intervals until 24 h. The concentrations of IgG, IgM, and IgA were lowest (p < 0.01) at birth and increased following colostrum feeding irrespective of the experimental group. Highest concentrations of all the Ig classes were observed at 12-24 h after birth. The concentrations then transiently decreased from day 7 to 14, and then steadily increased after day 28. The concentrations of IgG (p < 0.01) and IgA (p < 0.05) were higher in G-1 in relation to the other groups during the first week after birth. Similarly, higher concentration of IgA (p < 0.05) was found in G-1 in relation to the other groups during the rest of the experimental period. The apparent absorption efficiency of colostral IgG was higher (p < 0.05) in G-4 in relation to G-3. Growth rate and health status were not influenced by the method and time of first colostrum feeding. In conclusion, a 6-h delay in the first colostrum feeding reduced the level of serum Ig noticeably.     

Serological evidence of Neospora caninum infection in mithun (Bos frontalis) from India

Studies conducted on mithuns maintained at National Research Centre on mithun, Indian Council of Agricultural Research (ICAR), Nagaland, India and mithuns found in free-ranging condition of Nagaland revealed that the overall prevalence of antibodies to Neospora caninum in mithun was 10% (95% CI=5-15) when detected by a commercially available competitive enzyme-linked immunosorbent assay test. Highest (prevalence rate=16, 95% CI=8-24) seroprevalence was found in mithuns above 3 years of age and lowest (prevalence rate=2, 95% CI=0-6) in mithuns of 2-12 months old. No statistically significant difference was observed between male (prevalence rate=7, 95% CI=0-14) and female (prevalence rate=12, 95% CI=6-18) seroprevalences. The seroprevalence was found to be higher (prevalence rate=20, 95% CI=9-31) in mithuns found in free-ranging condition in comparison to mithuns kept in semi-intensive system (prevalence rate=5, 95% CI=1-9). This is probably the first report on serological evidence of N. caninum infection in mithun. The possible role of sylvatic fauna in the epidemiology of N. caninum infection mithun is also discussed in brief.     

Genetic characterisation of the mithun (Bos frontalis) and studies of spermatogenesis, blood groups and haemoglobins of its hybrids with Bos indicus

The mithun (Bos frontalis) and its hybrids with Bos indicus were studied to provide further cytogenetic information which might throw light on the mechanisms of the male hybrid infertility and facilitate the establishment of a stable crossbreed. It was shown that compared with conventional cattle the mithun has a homozygous, species specific 2/27 centric fusion which reduced the diploid chromosome number from 60 to 58. This provided further proof that Robertson translocation-type rearrangements have been the major source of interspecies karyotype differences in the evolution of the Bovidae. In the mithun there was also significant polymorphism between centromeres of non-homologous chromosomes and there was heteromorphism between several homologous chromosomes which could possibly serve as useful genetic markers for breeding programmes. In F1 hybrids spermatogenesis progressed to a relatively advanced stage, without going so far as to produce spermatozoa. In back crosses to B indicus spermatogenesis progressed further so that spermatozoa could be seen, though not as numerous as in normal bulls. In most hybrids there were haemoglobin bands which corresponded either to Hb A or Hb B of cattle but were much wider. It was shown that these were a combination of Hb Mi derived from the mithun and Hb A or Hb B derived from B indicus. In a few hybrids there were only Hb Mi. In these cases Hb Mi had been present in both parents and proved that the dam was not a pure siri. The possible mechanism of hybrid male infertility is discussed including faults in the epistatic gene effect between chromosomes and changes in the degree of association of centromeric regions in interspecies hybrids. It is suggested that additional cytogenetic examination of blood lymphocytes and especially of testicles would help the understanding of the fertility barriers of hybrid males and would make a breeding programme for a stable crossbreed possible.     

Comparative nutrient utilization,growth, and rumen enzyme profile of mithun (Bos frontalis) and Tho-tho cattle (Bos indicus) fed on tree-leaves-based ration

A study was conducted to compare the nutrient utilization, growth, and rumen enzyme profile of mithun (Bos frontalis) and Tho-tho cattle (Bos indicus) reared in the same feeding and managemental conditions. For the purpose, male mithun (n = 8) and male Tho-tho cattle (n = 8) of 1.5 years age, selected from the farm of National Research Centre on Mithun, Nagaland, India, were fed on mixed-tree-leaves-based ration as per the requirement of NRC (2001) for cattle for 12 months. Average daily gain (ADG), average dry matter intake (DMI), and feed conversion ratio (FCR) for all animals were recorded. A metabolic trial was conducted at 6 months of the experiment to assess the digestibility coefficient of different nutrients and nutritive value of ration. At 12 months of the experiment, rumen liquor was collected from all animals and analyzed for rumen enzyme profiles, viz., carboxymethylcellulase, xylanase, α-amylase, β-glucosidase, α-glucosidase, urease, and protease. It was found that ADG (507.8 g vs 392.8 g), DM intake (6.59 vs 5.85 kg/day) and DMI/W^0.75 (98.75 g vs 91.00 g/day), crude protein intake (780 vs 700 g/day), and total digestible nutrient intake (3.65 vs 3.32 kg/day) were higher (p < 0.05) in mithun than cattle. The nitrogen balance was higher and FCR was better (p < 0.05) in mithun compared with cattle. The digestibility coefficient of different nutrients was similar (p > 0.05) between the species. The microbial enzyme profiles of mithun and cattle were not different (p > 0.05). The better growth performance of mithun than cattle as found in the present study clearly indicates that the mithun has higher genetic potential for growth than Tho-tho cattle of north-eastern hilly region of India.     

Determination of effective dosage of GH-releasing factor for blood GH responses in mithun (Bos frontalis)

A study was undertaken to determine the effective dosage of GH-releasing hormone (GRF) required to produce blood GH response in mithun (Bos frontalis), a semi-wild ruminant species. For the purpose, 12 mithuns averaging 11.5 months of age and 146 kg body weight (BW) were randomly assigned to receive GRF (n = 12), administered at 0 (normal saline), 5, 10 and 20 mug per 100 kg BW. Blood samples were collected prior to and after GRF administration at -60, -45, -30, -15, -10, -5, 0 min and 5, 10, 15, 30 and thereafter, at 15-min interval up to 8 h post-GRF were assayed for plasma GH. For all the dosages, the pre-treatment GH concentrations and corresponding area under GH response curve (AUC) were similar (p > 0.05). The post-GRF plasma GH responses to different dosages of GRF viz. 5, 10 and 20 mug per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those recorded in normal saline-treated controls. The GH responses to 10 and 20 mug GRF per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those registered in mithuns administered with 5 mug GRF per 100 kg BW. Interestingly, post-GRF concentration of plasma GH and AUCs were not different for 10 and 20 mug GRF per 100 kg BW dosages. In all animals treated with GRF, a peak of GH was registered within 10 to 20 min post-GRF. Following 5 mug GRF per 100 kg BW, GH concentrations were maintained at higher level for 90 min post-GRF and thereafter became similar to that of controls and it was 435 min for 10 and 20 mug GRF per 100 kg BW dosages. In conclusion, our results suggest that 10 mug GRF per 100 kg BW is the dosage, which can be used for augmentation of mithun production.     

Detection of Shiga Toxin‐Producing Escherichia coli and Enteropathogenic Escherichia coli in Faecal Samples of Healthy Mithun (Bos frontalis) by Multiplex Polymerase Chain Reaction

Faecal samples obtained from 190 healthy mithuns were examined for the presence of Escherichia coli. Total one‐hundred and five E. coli isolates were obtained from these samples, which belonged to 55 different serogroups. These isolates were subjected to multiplex polymerase chain reaction (m‐PCR) for detection of stx1, stx2, eaeA and hlyA genes. Twenty‐three (21.90%) E. coli isolates belonging to 14 serogroups revealed the presence of at least one virulence gene when examined by m‐PCR. Nineteen percent and 2.85% of the mithuns were found to carry Shiga toxin‐producing E. coli (STEC) and enteropathogenic E. coli, respectively. stx1 and stx2 genes were found to be prevalent in 7 (6.67%) and 18 (17.14%) of the isolates respectively, whereas eaeA and hlyA genes were found to be carried by three (2.85% each) isolates. Interestingly, none of the STEC isolates belonged to serogroup O157.     

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post‐thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%–1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing–thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%–0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.     

Molecular phylogeny and diversity of Myanmar and Bhutan mithun based on mtDNA sequences

The mithun (Bos frontalis), synonymous with mithan and gayal, is considered to be a domesticated form of gaur (B. gaurus). However, there has been a controversy concerning its origin. In an effort to address this issue, the mitochondrial cytochrome b (cytb) genes of 20 mithun from Myanmar and 13 from Bhutan were sequenced to trace its maternal origin. Seven cytb haplotypes were found in the 33 mithun, and the phylogenetic tree for these haplotypes clearly showed three embranchments involving five gaur types, a B. indicus type, and a B. taurus type. Sixteen Myanmar and 12 Bhutan mithun had gaur haplotypes, while a B. indicus haplotype was found in three Myanmar and one Bhutan mithun. The B. taurus haplotype was detected in a single Myanmar animal. These results demonstrated that the principal maternal origin of mithun was gaur and suggested that it was directly domesticated from gaur. However, some introgression of domestic cattle existed in current mithun populations. The presence of cattle mtDNA raised the question of how many cattle nuclear genes might have been integrated into the gene pool of mithun.     

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A‐F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at ?196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Effect of pasteurized egg yolk on the quality of cryopreserved boar semen

Egg yolk (EY, control) is an essential ingredient of diluents for boar semen cryopreservation. Pasteurized egg yolk (PEY) reduces hygienic risks in processing and is easier to standardize. The aim of this study was to evaluate the in vitro effect of PEY (treatment) on frozen-thawed boar semen. In a split-sample approach (n = 13 boars), it could be shown that there is neither an influence (p > .05) on post-thawing motility (PTM: 5, 30 and 120 min) nor on morphologically intact sperm, percentage of acrosome defects and membrane fluidity using a PEY extender compared to the control. Mitochondrial activity (p = .043), membrane integrity (p = .015) and PTM 300 min (p = .023) were slightly affected in the treatment group. Overall, sperm quality was at a high level in both experimental groups. Further studies are needed to determine the impact of PEY on the fertilizing capacity of boar ejaculates.     

Karyotyping, red blood cell and haemoglobin typing of the mithun (Bos frontalis), its wild ancestor and its hybrids

In Bhutan in remote parts of the Himalayas the mithun (Bos frontalis) has been used probably for centuries in an ingenious system for crossbreeding with domestic cattle which results in highly profitable hybrid females. The hybrid males are infertile and so far no stable crossbreed has been developed. Genetic analyses reported in this paper support the view that the gaur is the wild ancestor of the mithun. Both have only 58 chromosomes in contrast to 60 in cattle and also different, hitherto undescribed haemoglobins and blood groups which would justify the revision of the present classification of the subfamily Bovinae. If, with the aid of modern genetic methods, a stable crossbreed could be developed, dairy and beef production in Bhutan and many climatically similar areas could benefit greatly. Because of its remarkable size the mithun may, in the hands of enterprising breeders, also make a useful genetic contribution to beef production elsewhere.     

Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.     

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Background

Proximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.

Methods

Three ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).

Results

Semen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).

Conclusion

Higher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.     

不同浓度防冻剂和投液氮温度对牛体外受精胚胎冷冻效果的影响

比较了3种不同浓度的甘油（1.4,1.0,0.7mol/L）和投液氮温度（-25℃、-30℃和-35℃）对牛体外受精胚胎冷冻效果的影响。结果发现,最终投液氮温度为-25℃时,胚胎冻后存活率以1.4mol/L甘油组为最高,同1.0mol/L甘油组相比,差异显著（78.5%vs 64.5%,P〈0.05）;与0.7mol/L甘油组相比,差异极显著（78.5%vs53.5%,P〈0.01）。以1.0mol/L的甘油冷冻,-30℃投液氮的效果优于-25℃的,它们之间冻后胚胎的孵化率差异显著（77.1%vs 64.5%,P〈0.05）。用0.7mol/L甘油冷冻,投液氮温度以-30℃的为最好,冻后胚胎的孵化率（73.3%）极显著（P〈0.01）高于-25℃的（53.5%）。用1.4mol/L甘油冷冻,以-25℃投液氮为最好,冻后胚胎的孵化率（78.5%）显著（P〈0.05）高于-35℃的（65.7%）。结果表明,不同的甘油浓度和投液氮温度对牛体外受精胚胎的冷冻效果有显著的影响。