Our objective was to determine effects of genetic potential for residual feed intake (RFI) and pre-natal diet on post-natal growth and reproductive development of bulls. Twenty-five bull calves with a divergent potential for RFI were produced by mating purebred Angus heifers (60) categorised as high or low RFI and inseminated with semen from high or low RFI bulls. From 30 to 150 days of gestation, half the heifers (balanced for RFI across treatments) in each RFI category were fed to gain 0.7 or 0.5 kg/day (~100% and 75% of recommended feed intake; designated normal- and low-diet, respectively). Birthweights of bull calves (n = 25) did not differ significantly among treatment groups (P ≥ 0.1). However, there was a tendency (P < 0.1) for a maternal diet*time interaction for bull weights, with bulls from low pre-natal diet fed heifers growing faster between 10 and 16 months of age than bulls from normal pre-natal diet fed heifers. Furthermore, high-RFI bulls had a larger scrotal circumference (P < 0.01) and attained puberty (P < 0.05) earlier than low-RFI bulls. Other testes measures, including paired testes volume, epididymal sperm reserves, daily sperm production and total sperm production, were not significantly different among treatment groups (P ≥ 0.1). High RFI bulls tended (P < 0.1) to have better average sperm motility than low RFI bulls. However, sperm morphology, viability and chromatin damage were not different (P ≥ 0.1). In summary, young beef bulls with low RFI had smaller testis, delayed onset of puberty (~1.7 months) and tended to exhibit lower progressive motility compared with high RFI bulls. Pre-natal diet had no direct influence on male reproductive development, nor did it interact with RFI of bulls.

Well-fed prepuberal Holstein bulls had larger testes, earlier puberty, higher LH, testosterone and IGF-1, earlier and more proliferating and differentiating Sertoli cells, and greater sperm production potential. The objective was to determine effects of pre-pubertal nutrition on mRNA expression of testicular genes. Holstein bull calves were fed high or low diets (20 or 12% crude protein, respectively and 71.6 or 64.4% Total Digestible Nutrients) from 2 wk, castrated at 8, 16, 24 and 32 wk and testicular mRNA extracted and sequenced. Differential expression of genes mainly occurred at 16 and 24 wk. At 16 wk, functional analysis (DAVID) of DE mRNA revealed common biological processes including “cholesterol” and “fatty acid biosynthesis,” with most genes (including HMGCR, HMGCS1, HSD17) upregulated in high-diet bulls (P < 0.05). Major pathways enriched at 16 wk were “cholesterol biosynthesis”, “steroid metabolism” and “activation of gene expression by Sterol regulatory element binding protein (SREBP)” (P < 0.05). In high-diet bulls, mature Sertoli cell marker Connexin 43, was upregulated at 16 wk and immature Sertoli cell marker (AMH) downregulated at 24 wk. There was an indirect interaction between insulin family receptor and most upregulated cholesterol biosynthesis genes. Pre-pubertal nutrition enhanced testicular cholesterol/steroid biosynthesis and Sertoli cell maturation.

The critical role of insulin-like growth factor (IGF) 1 in promoting Sertoli cell proliferation invivo and invitro has been established, but its downstream signalling mechanisms remain unknown. In addition to mitogenic effects, a role for IGF1 in mediating cholesterol biosynthesis within testes has been implied. The aims of this study were to investigate the roles of: (1) phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signalling in IGF1-mediated Sertoli cell proliferation; and (2) IGF1 in mediating cholesterol biosynthesis in Sertoli cells. Primary cultures of Sertoli cells were prepared from 1-week-old porcine testes. On Day 3 of culture, Sertoli cells were treated with 300ng mL−1 IGF1, alone or in combination with inhibitors of IGF1 receptor (2μM picropodophyllotoxin), Akt (1μM wortmannin) or mTOR (200nM rapamycin). Cells were cultured for 30min and phosphorylation levels of Akt, mTOR and p70 ribosomal protein S6 kinase (p70S6K) were determined by immunoblotting. Cell proliferation and quantitative polymerase chain reaction assays were conducted using cells cultured for 24h. IGF1 increased phosphorylation of Akt, mTOR and p70S6K and cell proliferation, and these effects were inhibited by inhibitors of IGF1R, Akt and mTOR. Furthermore, IGF1 upregulated the expression of cholesterol biosynthetic genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) and cytochrome P450, family 5, subfamily A, polypeptide 1 (CYP5A1)), but not sterol regulatory element-binding transcription factor 1 (SREBF1). Increased phosphorylation of p70S6K, a major downstream target of mTOR, and upregulated expression of genes involved in cholesterol biosynthesis are indicative of the key role played by IGF1 in regulating the synthesis of cholesterol, the precursor for steroid hormones.

Supplemental energy and protein during calf-hood (2-30 wk) in dairy bulls hastened puberty (~1 mo), upregulated steroid biosynthesis, concentrations of reproductive hormones and Sertoli cell maturation, with larger testes and greater sperm production (~25%) in mature bulls. The objective was to evaluate effects of feeding high (20.0% crude protein [CP], 67.9% total digestible nutrients [TDN]), control/ medium (17.0% CP, 66.0% TDN) and low (12.2% CP, 62.9% TDN) diets from 2 to 30 wk on post-pubertal testes of Holstein bulls. Based on RNA sequencing, 497 and 2961 genes were differentially expressed (p < 0.1) in high-vs low-and high-vs medium-diet groups, respectively. According to KEGG analysis, oxidative phosphorylation and ribosome pathways were upregulated in high-vs medium-and low-diet groups, with majority of upregulated genes encoding for essential subunits of complex I, III, IV and V of OXYPHOS pathway. In addition, mitochondrial translation, mitotic nuclear division and cell division were enriched in high-vs medium-diet groups. Consistent with these results, a greater percentage of sperm from high-diet bulls were progressively motile and had normal mitochondrial function compared to medium-diet sperm (p < 0.1). Thus, enhanced early life nutrition upregulated mitochondrial function in testes and sperm of post-pubertal Holstein bulls.Enhanced early-life nutrition hastens puberty, with ~25% increases in testis size and sperm production in dairy and beef bulls 1,2 . The underlying endocrine basis is profound increases in luteinizing hormone (LH) pulse frequency and insulin like growth factor (IGF-I) concentrations in blood 3,4 . In a cohort-based study in Sweden, low nutrition during the pre-pubertal period in men reduced susceptibility to heart disease in their offspring 5 , consistent with developmental programming in male germ cells that extends well into early post-natal life 6 . Therefore, nutritional modulation during early life affects testicular development, with apparent epigenetic effects on post-pubertal sperm function.We reported increased cholesterol/steroid biosynthesis and Sertoli cell maturation in testicular tissues of high-diet bulls at 16 and 24 wk, respectively 7 . Although a wide range in pre-pubertal diets had no significant effects on routine analyses of sperm morphology and function 8 , molecular-level analyses should be done, to ensure no deleterious effects are transmitted to the next generation. Our objective was to determine effects of pre-pubertal dietary modulations on post-pubertal testes of Holstein bulls. ResultsmRNA profile of testes. Approximately 340 × 10 6 reads were obtained from the 24 libraries sequenced. On average, 14,357,890 (SD = 2,267,076) reads were obtained per library and 13,565,597 (SD = 2,148,261) mapped to the Ensemble gene annotation database. On average 13,710,936 (SD = 3,098,630), 13,120,341 (SD = 1,219,498) and 13,865,513 (SD = 1,929,109) reads were mapped from HD, MD and LD groups respectively. A total of 18,288 genes were detected in testicular tissue and...

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Context Selection for residual feed intake (RFI) and its impact on male reproductive development has had mixed reviews in the past. Our previous studies demonstrated earlier puberty, larger testes and greater percentage of progressively motile sperm in high-RFI bulls. However, the molecular mechanisms within testes of bulls with varying RFI remain unclear. Aims To determine the effect of RFI and pre-natal diet on the expression patterns of testicular genes and use this information to explain differences observed across RFI. Methods The study included 25 purebred-Angus bulls with a genetic background of either high or low RFI and fed either normal or low pre-natal nutrition from 30 to 150 days post conception. After slaughter (17 months), testicular tissue was recovered, and RNA was extracted and sequenced. Key results Of 19218 expressed genes, 17 were differentially expressed for RFI (including PLCD1, INPP4B), with no differences being observed for pre-natal diet or diet × RFI interaction (false discovery rate) &lt; 0.1%). KEGG pathway analysis indicated that differentially expressed genes were associated with inositol phosphate metabolism, and phosphatidylinositol signalling. On the basis of a candidate gene-expression study, IGF1R was upregulated in high-RFI bulls (P &lt; 0.1). Conclusions Increased expression of IGF1R and lowered PLCD1 and INPP4B expression could activate PI3K–Akt signalling responsible for cell growth, proliferation and steroid metabolism in high-RFI bulls. Implications Selecting bulls for feed efficiency might affect molecular networks associated with reproduction and fertility.

Sperm cryopreservation and thawing reduces fertility and alters the content and function of various sperm proteins. Previously, we reported that a testes-specific isoform of angiotensin-converting enzyme (tACE) was required for capacitation of bovine spermatozoa. The aim of the present study was to determine effects of sperm cryopreservation and thawing on the content, activity and localisation of tACE in bovine spermatozoa. Relative median fluorescence intensity (flow cytometry) was greater (P<0.01), tACE content (110 kDa protein) in sperm proteins was higher (P<0.01) and there was greater tACE enzyme activity (mean (±s.e.m.) 0.16±0.01 vs 0.06±0.02UmL-1; P<0.01) in fresh versus frozen-thawed spermatozoa (n=6 bulls). In fresh spermatozoa, tACE was immunolocalised in the acrosomal and principal piece regions of the sperm head and tail respectively. However, in frozen-thawed spermatozoa, there were four patterns of localisation: most frozen-thawed spermatozoa (64%) had fluorescence in the acrosomal ridge, whereas in 17% and 9% of spermatozoa the signal was limited to the post-acrosomal region and the equatorial segment respectively; in the remainder (10%), there was no signal. We conclude that cryopreservation and thawing decrease the content and activity of tACE and cause it to be translocated to other parts of the sperm head.

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