A.M. English

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation.

Genomic identification, expression profiling, and functional characterization of CatSper channels in the bovine†

Abstract Cation channels of sperm (CatSper) are sperm-specific calcium channels with identified roles in the regulation of sperm function in humans, mice, and horses. We sought to employ a comparative genomics approach to identify conserved CATSPER genes in the bovine genome, and profile their expression in reproductive tissue. We hypothesized that CATSPER proteins expressed in bull testicular tissue mediates sperm hyperactivation and their rheotactic response in the reproductive tract of the cow. Bioinformatic analysis identified all four known CATSPER genes (CATSPER 1–4) in the bovine genome, and profiling by quantitative real-time polymerase chain reaction identified site-specific variation in messenger ribonucleic acid (mRNA) expression for all four genes along the reproductive tract of the bull. Using a novel antibody against CATSPER 1, protein expression was confirmed and localized to the principal piece of bull sperm, in agreement with what has been reported in other species. Subsequent treatment of bull sperm with either the calcium chelator ethylene glycol tetraacetic acid;mibefradil, a specific blocker of CatSper channels in human sperm; or CATSPER1 antibody all significantly inhibited caffeine-induced hyperactivation and the rheotactic response, supporting the concept that the calcium influx occurs via CatSper channels. Taken together, the work here provides novel insights into expression and function of CatSper channels in bull testicular tissue and in the function of ejaculated sperm. Summary Sentence The effect of blocking calcium channels on hyperactivation and rheotactic response.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: II. Effects on metabolic and reproductive endocrinology and identification of physiological markers of puberty and sexual maturation

The aim of this study was (1) to examine the effect of plane of nutrition during the first and second 6 mo of life on systemic concentrations of reproductive hormones and metabolites in Holstein-Friesian dairy bulls, and (2) to establish relationships with age at puberty and postpubertal semen production potential. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high or low plane of nutrition for the first 6 mo of life. At 24 wk of age, bulls were reassigned, within treatment, either to remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: high-high, high-low, low-low, and low-high. Monthly blood samples were analyzed for metabolites (albumin, urea, total protein, β-hydroxybutyrate, glucose, nonesterified fatty acid, triglycerides and creatinine), insulin, insulin-like growth factor-1, leptin, adiponectin, FSH, and testosterone. A GnRH challenge was carried out at 16 and 32 wk of age (n = 9 bulls per treatment). Blood was collected at 15-min intervals for 165 min, with GnRH administered (0.05 mg/kg of body weight, i.v.) immediately after the third blood sample. Blood samples were subsequently analyzed for LH, FSH, and testosterone. Stepwise regression was used to detect growth and blood measurements to identify putative predictors of age at puberty and subsequent semen quality traits. Metabolic hormones and metabolites, in general, reflected metabolic status of bulls. Although FSH was unaffected by diet, it decreased with age both in monthly samples and following GnRH administration. Testosterone was greater in bulls on the high diet before and after 6 mo of age. Testosterone concentrations increased dramatically after 6 mo of age. Luteinizing hormone was unaffected by diet following GnRH administration but basal serum LH was greater in bulls on a high diet before 6 mo of age. In conclusion, the plane of nutrition offered before 6 mo of age influenced metabolic profiles, which are important for promoting GnRH pulsatility, in young bulls.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: I. Effects on performance, body composition, age at puberty, and postpubertal semen production

The aim of this study was to examine the effect of plane of nutrition (1) during the first 6 mo of life and (2) from 6 mo of age to puberty on early growth characteristics, age at puberty, and postpubertal semen production in Holstein-Friesian bulls. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high (Hi) or low (Lo) plane of nutrition for the first 6 mo of life. The Hi and Lo calves received 1,200 and 450 g of milk replacer, respectively; Hi calves were fed concentrate ad libitum and Lo were fed a maximum of 1 kg concentrate daily, and concentrate allowances remained the same after weaning. At 24 wk of age, bulls were reassigned within treatment to either remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: Hi-Hi, Hi-Lo, Lo-Lo, and Lo-Hi. After puberty, all bulls were fed a moderate plane of nutrition until 60 wk of age; thereafter, the diet was ad libitum concentrates until slaughter at 72 wk of age. Bulls were weighed weekly before weaning and every 2 wk after weaning. Scrotal circumference (SC) was measured every 2 wk, beginning at 15 wk of age. Beginning at a SC of 24 cm, electro-ejaculation was carried out every 2 wk to establish the onset of puberty. Semen collection continued monthly after puberty. Thermal images of the scrotum were taken monthly from 28 to 36 wk of age. Scrotal skin thickness (SST) was measured monthly (from 16 wk of age to puberty) using a digital calipers. Bulls on the Hi diet had a higher scrotal temperature and SST at each time point than those on the Lo diet. Average daily gain (ADG) was greatest in Hi-Hi bulls, with Hi-Lo and Lo-Hi having similar ADG but both being greater than Lo-Lo. Bulls on the Hi diet pre-6 mo of age were younger at puberty, regardless of diet offered post-6 mo of age. Bulls offered a Hi diet post-6 mo were heavier at puberty. Neither scrotal temperature nor dietary treatment affected postpubertal semen production variables. In conclusion, a high plane of nutrition during the first 6 mo of age hastened the onset of puberty and the availability of saleable semen, regardless of plane of nutrition post-6 mo of age.