Janivara Parameswaraiah Ravindra

Evaluation of maize grain and polyunsaturated fatty acid (PUFA) as energy sources for breeding rams based on hormonal, sperm functional parameters and fertility

The objective of the present study was to elucidate the effect of different sources of dietary energy (maize vs polyunsaturated fatty acid (PUFA) on semen functional parameters and fertility of adult rams. Eighteen adult rams were divided into two groups (maize and PUFA, n=9). The main energy source for the rams in the maize group was coarsely ground maize grain, whereas in the PUFA group it was sunflower oil (rich in 18:2 linoleic acid, an omega-6 acid). The ration was fed for a minimum period of 60 days and thereafter semen was collected for evaluation. The proportion of progressive forward motility was significantly (P<0.05) higher in the PUFA group compared with the maize group. Sperm lipid peroxidation as measured by malondialdehyde formation (µM per 1×10(9) spermatozoa) was significantly (P<0.05) higher in the PUFA group compared with the maize group. When the semen was diluted with Tris-egg yolk-citrate buffer and incubated for 24h at 4°C, the proportions of plasmalemma integrity, the sperm subpopulation positive for functional membrane and acrosomal integrities, and mitochondrial membrane potential were significantly (P<0.05) higher in PUFA-fed than in maize-fed animals. The different sources of energy did not influence the serum and seminal plasma IGF-I levels. The cleavage rate (percentage) did not differ significantly between PUFA- (45.4±4.91) and maize- (44.63±6.8) fed animals. In conclusion, PUFA feeding influenced sperm quality by altering or stabilising membrane integrity. The present study indicates that PUFA may improve semen quality but did not improve in vitro fertilisation.

Effect of dietary energy on seminal plasma insulin-like growth factor-I (IGF-I), serum IGF-I and testosterone levels, semen quality and fertility in adult rams.

The objective of the present study was to modulate seminal plasma insulin-like growth factor-I (IGF-I) by dietary energy and assess the relationship among testosterone and IGF-I levels, semen quality and fertility in adult rams. Twenty-four 1-yr old adult Nellore rams were equally divided into three groups (n = 8) and fed with three different concentrate mixtures formulated using conventional ingredients and finger millet (Eleucine corocana) straw to ensure rams received with similar amount of crude protein with three levels of energy. Rams in low-energy group were offered diets with 20% less energy than the control energy group (optimum energy, 100%, recommended energy level), whereas rams in high energy group were offered diets with 20% more energy than the optimum energy group. Semen was collected from rams 60 days after start of the experimental feeding. The percentages of progressive forward motility, functional membrane integrity and mitochondrial membrane potential of the spermatozoa were significantly (P < 0.05) higher in control and high energy groups as compared to low-energy group. Feeding of low-energy diet significantly (P < 0.05) decreased spermatozoa VSL, VCL and VAP when compared to control and high energy fed groups. The number of spermatozoa binding/oocyte was significantly (P < 0.05) higher in control (11.23 ± 0.20) and high energy (10.57 ± 0.19) groups as compared to the low energy (6.14 ± 0.01) group. The serum and seminal plasma IGF-I levels were significantly (P < 0.05) higher in control and high energy fed groups as compared to the low-energy group. The serum testosterone and cholesterol levels were significantly (P < 0.05) higher in the control group as compared to the low-energy group. The seminal plasma fructose levels in optimum energy fed animals were significantly (P < 0.05) higher as compared to other two groups. The seminal plasma IGF-I level had positive correlation with progressive forward motility (r = 0.7) and other velocity (linearity, r = 0.7; straightness, r = 0.7) parameters. The study suggested that the modulation of seminal plasma IGF-I levels by dietary energy is possible and the optimum level of seminal plasma IGF-I is necessary and sufficient to influence semen quality.

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility

Abstract The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (nu2009=u200912) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (nu2009=u20093) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1–5.6, 37%) and basic (pI; 7.9–10.0, 27%) proteins and their pattern of expression varied significantly (pu2009<u20090.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4u2009kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (pu2009<u20090.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus).

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.

Spermatozoal transcripts expression levels are predictive of semen quality and conception rate in bulls (Bos taurus)

Spermatozoal transcripts expression levels could be used to assess fertility potential of a male. The objective of the present study was to elucidate the predictive ability of the expression levels of growth, apoptosis and homeostasis regulating transcripts on sperm functions and fertility. The expression levels of spermatozoal RNA isolated from the neat semen samples were related to the good (discarded ejaculate, <25%; nxa0=xa07) and poor (discarded ejaculate, >40%, nxa0=xa06) quality semen producer and bulls (nxa0=xa012) with known conception rate. The relative fold expression levels of BMP2 were significantly (pxa0<xa00.01) higher in good than the poor semen producers and positively associated with post-thaw sperm velocity parameters (LIN and VAP). The NGF expressions fold levels had significant (pxa0<xa00.05) positive relationship with mitochondrial membrane potential of neat semen samples. The genes involved in the apoptotic, UBE2D3 (rxa0=xa0-0.61, pxa0=xa00.02), CASP3 (rxa0=xa0-0.57, pxa0=xa00.03) and homeostatic, HSFY2 (rxa0=xa0-0.61, pxa0<xa00.02) regulators had significant negative correlation with the percentage of post-thaw fast progressive motile spermatozoa. The expression level of TRADD had significant negative influence on the mitochondrial membrane potential (rxa0=xa0-0.54, pxa0=xa00.05) of neat semen samples and conception rate (rxa0=xa0-0.57, pxa0<xa00.05). The expression levels of BMP2 had highly significant positive correlation with NGF (rxa0=xa00.99, pxa0<xa00.01) and CASP3 (rxa0=xa00.56, pxa0=xa00.05). The BMP2 expression level might be used to predict the quality of the semen and TRADD determine the conception rate of the bull. The study provides ample evidence that the sperm transcripts expression levels might be used to predict quality semen production and bull fertility.

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.

Relationship between seminal plasma tuberoinfundibular peptide of 39 residues and sperm functional attributes in buffalo (Bubalus bubalis).

The buffalo seminal plasma protein profile and its relationship with sperm quality have not been studied in detail. Thus, the aim of the present study was to profile buffalo seminal plasma proteins and to assess the relationship between differentially expressed proteins and sperm characteristics. Semen samples (n = 44) were collected from 11 Murrah buffalo bulls (four ejaculates from each animal) and seminal plasma protein profiling was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionisation time-of-flight analysis of one of the differentially expressed proteins, namely the 11-12 kDa protein, identified it as tuberoinfundibular peptide of 39 residues (TIP39). Western blot analysis confirmed the presence of TIP39, with TIP39 expression in seminal plasma varying among bulls. Based on TIP39 levels, bulls were classified into two groups, those with high and low protein. The percentages of spermatozoa positive for mitochondrial membrane potential test, chromatin distribution test, synthetic media sperm penetrability test and acrosomal integrity test were significantly (P < 0.05) high in the high protein group. The present study is the first to demonstrate the presence of TIP39 in buffalo seminal plasma and the positive effect of TIP39 on the functional parameters and fertilising ability of spermatozoa.

Protective role of epigallocatechin-3-gallate on arsenic induced testicular toxicity in Swiss albino mice

Arsenic, often referred to as the king of poisons is carcinogenic in humans and animals. It affects multiorgan systems including reproduction. The present study was undertaken to explore the protective role of green tea compound, epigallocatechin-3-gallate (EGCG) on arsenic induced testicular toxicity in Swiss albino mice. Thirty two adult male mice were randomly assigned to four groups (n=8). Group I served as control without test chemical. The group II received arsenic (200ppm) through drinking water, group III received only EGCG (20mg/kgb.wt., intraperitoneally, alternate days) and group IV was administered arsenic+EGCG for 40days. Factorial experimental design was employed to assess the treatment effect. The EGCG restored arsenic induced decrements in epididymal sperm concentration, kinematic attributes (total motility, rapid, progressive motile, fast progressive, VSL, VAP, VCL, BCF, LIN, WOB, STR and Type A), structutal membrane integrity, functional membrane integrity and mitochondrial membrane potential. As evidenced by the histoarchitectural studies, the EGCG reversed the deleterious effects of arsenic on testicular malondialdehyde (p<0.05) levels, reduced glutathione, antioxidative enzymes and spermatogenesis. Overall, the results suggest that EGCG reduces the testicular oxidative stress induced by arsenic poisoning and thereby protect the reproductive system.

Effect of Different Diet Level on the Physiological Adaptability, Biochemical and Endocrine Responses and Relative Hepatic HSP70 and HSP90 Genes Expression in Osmanabadi Kids

The study was conducted to investigate the impact of different levels of feed on the adaptive capability based on physiological, blood biochemical, endocrine and molecular mechanisms in growing Osmanabadi kids. The primary objective of the study was to identify if HSP70 and HSP90 can be a nutritional stress marker for goat. The study was conducted for a period of two months. The animals were randomly divided into three groups as GI (n = 6; ad libitum feeding), GII (n = 6; 20% less than ad libitum) and GIII (n = 6; 40% less than ad libitum). The animals were fed with feed consisting of 50% roughage and 50% concentrate. Blood collection was carried out at fortnightly intervals. Body weights were recorded at weekly interval. Physiological responses, biochemical responses, plasma tri-iodo-thyronine (T3), thyroxin (T4) and cortisol were recorded at fortnightly interval. At the end of study period, only GI and GIII animals were slaughtered and different organs were collected for histopathological studies as well as for hepatic HSP70 and HSP90 mRNA transcript expression. Body weight recorded showed significant (P < 0.01) differences between the groups. Physiological responses showed significant (P < 0.01) variation among the groups. Among the biochemical parameters, plasma glucose and total plasma protein and globulin showed significant (P < 0.01) differences between the groups. Plasma T3 (P < 0.01), T4 (P < 0.01) and cortisol (P < 0.05) also differed significantly between the groups. The relative hepatic HSP70 mRNA transcript expression was significantly (P < 0.05) higher in GIII (2.8 fold) as compared to GI (1 fold) kids. Similar result was obtained for hepatic HSP90 mRNA transcript expression. From the results, it can be concluded that Osmanabadi kids possessed the ability to alter their adaptive mechanisms to maintain homeostasis. Further, the study revealed the significance of providing the optimum nutrition for these animals to adapt to existing environmental conditions. The study also established that respiration rate (RR), rectal temperature (RT), T3, T4 and cortisol are considered as nutritional stress markers for goat. Further, the results revealed that probably this is the first study to establish the nutritional stress impact on heat shock protein (HSP) expression in goats. The study identified both HSP70 and HSP90 to be the ideal molecular markers for feed deficit in goats.