Pre- and post-puberty expression of genes and proteins in the uterus of Bos indicus heifers: the luteal phase effect post-puberty.

Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre- and six post-pubertal Brahman heifers. Post-pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA-sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P-value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10-6 ). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 × 10-5 ) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post-puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567, ZNF775, RELA, PIAS2, LHX4, SOX2, MEF2C, ZNF354C, HMG20A, TCF7L2, ZNF420, HIC1, GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function.

Blastocyst-induced changes in the bovine endometrial transcriptome.

The objectives of this study were (i) to determine whether blastocyst-induced responses in endometrial explants were detectable after 6- or 24-h co-culture in vitro; (ii) to test if direct contact is required between embryos and the endometrial surface in order to stimulate endometrial gene expression; (iii) to establish the number of blastocysts required to elicit a detectable endometrial response; (iv) to investigate if upregulation of five interferon-stimulated genes (ISGs) in the endometrium was specific to the blastocyst stage and (v) to test if alterations in endometrial gene expression can be induced by blastocyst-conditioned medium. Exposure of endometrial explants to Day 8 blastocysts in vitro for 6 or 24 h induced the expression of ISGs (MX1, MX2, OAS1, ISG15, RSAD2); expression of IFNAR1, IFNAR2, NFKB1, IL1B, STAT1, LGALS3BP, LGALS9, HPGD, PTGES, ITGB1, AKR1C4, AMD1 and AQP4 was not affected. Culture of explants in the presence of more than five blastocysts was sufficient to induce the effect, with maximum expression of ISGs occurring in the presence of 20 blastocysts. This effect was exclusive to blastocyst stage embryos; oocytes, 2-cell embryos or Day 5 morulae did not alter the relative abundance of any of the transcripts examined. Direct contact between blastocysts and the endometrial surface was not required in order to alter the abundance of these transcripts and blastocyst-conditioned medium alone was sufficient to stimulate a response. Results support the notion that local embryo-maternal interaction may occur as early as Day 8 of pregnancy in cattle.

Morphological defects, sperm DNA integrity, and protamination of bovine spermatozoa.

The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n = 1009) from two-year-old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap-frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2  = 0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p < 0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2  = -0.168, MCB; -0.116 and HCB; -0.137 (p < 0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration.

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed-time artificial insemination protocol in Australian Bos indicus cattle.

Increasing use of fixed-time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2 ) in a Tris-egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post-thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.

Transcriptome analyses identify five transcription factors differentially expressed in the hypothalamus of post- versus prepubertal Brahman heifers.

Puberty onset is a developmental process influenced by genetic determinants, environment, and nutrition. Mutations and regulatory gene networks constitute the molecular basis for the genetic determinants of puberty onset. The emerging knowledge of these genetic determinants presents opportunities for innovation in the breeding of early pubertal cattle. This paper presents new data on hypothalamic gene expression related to puberty in (Brahman) in age- and weight-matched heifers. Six postpubertal heifers were compared with 6 prepubertal heifers using whole-genome RNA sequencing methodology for quantification of global gene expression in the hypothalamus. Five transcription factors (TF) with potential regulatory roles in the hypothalamus were identified in this experiment: , , , , and . These TF genes were significantly differentially expressed in the hypothalamus of postpubertal versus prepubertal heifers and were also identified as significant according to the applied regulatory impact factor metric ( < 0.05). Two of these 5 TF, and , were zinc fingers, belonging to a gene family previously reported to have a central regulatory role in mammalian puberty. The gene belongs to the family of homologues of Drosophila sine oculis () genes implicated in transcriptional regulation of gonadotrope gene expression. Tumor-related genes such as and are known to affect basic cellular processes that are relevant in both cancer and developmental processes. Mutations in were associated with puberty in humans. Mutations in these TF, together with other genetic determinants previously discovered, could be used in genomic selection to predict the genetic merit of cattle (i.e., the likelihood of the offspring presenting earlier than average puberty for Brahman). Knowledge of key mutations involved in genetic traits is an advantage for genomic prediction because it can increase its accuracy.

Seminal plasma proteins and their relationship with percentage of morphologically normal sperm in 2-year-old Brahman (Bos indicus) bulls.

The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (p<0.05). A multiple regression analyses of these SPPs, using three models; non-parametric Tree Model, Generalized Additive Model, and a step-wise selection method were conducted, and 6 SPPs could be used to predict PNS24; four SPPs had positive and two had negative association with PNS24. Together these spots explained 35% of the phenotypic variation in PNS24. Using mass spectrometry (MALDI-ToF and TripleToF-MS) the SPPs with positive relationship contained mainly apolipoprotein A-I (1310), protein DJ-1 and glutathione peroxidase 3 (2308), phosphoglycerate kinase 1 (6402) and apolipoprotein A-I and secretoglobin family 1D member (8008). The SPPs inversely associated with PNS24 were clusterin/seminal plasma protein A3 (1411) and epididymal secretory protein E1 (8108). This is the first comprehensive report on the association between seminal plasma protein composition in Bos indicus Brahman bulls and sperm morphology.

Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively.

A field investigation of a modified intravaginal progesterone releasing device and oestradiol benzoate based ovulation synchronisation protocol designed for fixed-time artificial insemination of Brahman heifers.

Pregnancy rates (PR) to fixed-time AI (FTAI) in Brahman heifers were compared after treatment with a traditional oestradiol-based protocol (OPO-8) or a modified protocol (OPO-6) where the duration of intravaginal progesterone releasing device (IPRD) was reduced from 8 to 6 days, and the interval from IPRD removal to oestradiol benzoate (ODB) was increased from 24 to 36 h. Rising 2 yo heifers on Farm A: (n = 238 and n = 215; two consecutive days AI); B (n = 271); and C (n = 393) were allocated to OPO-8 or OPO-6. An IPRD was inserted and 1mg ODB i.m. on Day 0 for OPO-8 heifers and Day 2 for OPO-6 heifers. On Day 8, the IPRD was removed and 500 µg cloprostenol i.m. At 24h, for OPO-8 heifers, and 36 h, for OPO-6 heifers, post IPRD removal all heifers received 1mg ODB i.m. FTAI was conducted at 54 and 72 h post IPRD removal for OPO-8 and OPO-6 heifers. At Farm A, OPO-6 heifers, AI on the second day, the PR was 52.4% to FTAI (P = 0.024) compared to 36.8% for OPO-8 heifers. However, no differences were found between OPO-8 and OPO-6 protocols at Farm A (first day of AI) (39.9 vs. 35.7%), or Farms B (26.2 vs. 35.4%) and C (43.2% vs. 40.3%). Presence of a corpus luteum at IPRD insertion affected PR to FTAI (43.9% vs. 28.8%; P < 0.001). This study has shown that the modified ovulation synchronisation protocol OPO-6 may be a viable alternative to the OPO-8 protocol for FTAI in B. indicus heifers.

Evaluation and histological examination of a Campylobacter fetus subsp. venerealis small animal infection model.

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0% to 100% and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion.

Seminal plasma proteome of electroejaculated Bos indicus bulls.

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4 ± 2.3 and 64 ± 3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70 kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 d-isomerase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase 1. In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.

Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls.

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility.

Follicle stimulating hormone secretion and dominant follicle growth during treatment of Bos indicus heifers with intra-vaginal progesterone releasing devices, oestradiol benzoate, equine chorionic gonadotrophin and prostaglandin F(2α).

The aim of this study was to investigate the effects on follicle stimulating hormone (FSH) secretion and dominant follicle (DF) growth, of treatment of Bos indicus heifers with different combinations of intra-vaginal progesterone releasing devices (IPRD), oestradiol benzoate (ODB), PGF2α and eCG. Two-year-old Brahman (BN; n=30) and Brahman-cross (BNX; n=34) heifers were randomly allocated to three IPRD-treatments: (i) standard-dose IPRD [CM 1.56g; 1.56g progesterone (P4); n=17]; (ii) half-dose IPRD (CM 0.78g; 0.78g P4; n=15); (iii) half-dose IPRD+300IU eCG at IPRD removal (CM 0.78g+G; n=14); and, (iv) non-IPRD control (2×PGF2α; n=18) 500µg cloprostenol on Days -16 and -2. IPRD-treated heifers received 250µg PGF2α at IPRD insertion (Day -10) and IPRD removal (Day -2) and 1mg ODB on Day -10 and Day -1. Follicular dynamics were monitored daily by trans-rectal ultrasonography from Day -10 to Day 1. Blood samples for determination of P4 were collected daily and samples for FSH determination were collected at 12h intervals from Day -9 to Day -2. A significant surge in concentrations of FSH was observed in the 2×PGF2α treatment 12h prior and 48h after follicular wave emergence, but not in the IPRD-treated heifers. Estimated mean concentrations of total plasma P4 during the 8 days of IPRD insertion was greater (P<0.001) in the CM 1.56g P4 treated heifers compared to the CM 0.78g P4 treated heifers (18.38ng/ml compared with 11.09ng/ml, respectively). A treatment by genotype interaction (P=0.036) was observed in the mean plasma P4 concentration in heifers with no CL during IPRD insertion, whereby BN heifers in the CM 1.56g treatment had greater plasma P4 than the BNX heifers on Days-9, -7, -6, -5, and -4. However, there was no genotype effect in the CM 0.78g±G or the 2×PGF2α treatment. Treatment had no effect on the DF growth from either day of wave emergence (P=0.378) or day of IPRD removal (P=0.780) to ovulation. This study demonstrates that FSH secretion in B. indicus heifers treated with a combination of IPRD's and ODB to synchronise ovulation was suppressed during the period of IPRD insertion but no significant effect on growth of the DF was observed.

The integrity of sperm chromatin in young tropical composite bulls.

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r=0.34, P=0.0076), Mot (r=0.36, P=0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r=0.31, P=0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r=-0.28, P=0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r=-0.53, P<0.0001), the percentage of sperm with head abnormalities (r=0.68, P<0.0001) and the percentage of intact sperm (Int) with SBH (r=-0.26, P=0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.

Animal-level factors affecting ovarian function in Bos indicus heifers treated to synchronize ovulation with intravaginal progesterone-releasing devices and oestradiol benzoate.

The primary objective of this study was to investigate the impact of animal-level factors including energy balance and environmental/management stress, on the ovarian function of Bos indicus heifers treated to synchronize ovulation. Two-year-old Brahman (BN) (n = 30) and BN-cross (n = 34) heifers were randomly allocated to three intravaginal progesterone-releasing device (IPRD) treatment groups: (i) standard-dose IPRD [Cue-Mate(®) (CM) 1.56 g; n = 17]; (ii) half-dose IPRD [0.78 g progesterone (P(4)); CM 0.78 g; n = 15]; (iii) half-dose IPRD + 300 IU equine chorionic gonadotrophin at IPRD removal (CM 0.78 g + G; n = 14); (iv) and a control group, 2× PGF(2α) [500 µg prostaglandin F(2α) (PGF(2α))] on Day -16 and -2 (n = 18). Intravaginal progesterone-releasing device-treated heifers received 250 µg PGF(2α) at IPRD insertion (Day -10) and IPRD removal (Day -2) and 1 mg oestradiol benzoate on Day -10 and -1. Heifers were managed in a small feedlot and fed a defined ration. Ovarian function was evaluated by ultrasonography and plasma P(4) throughout the synchronized and return cycles. Energy balance was evaluated using plasma insulin-like growth factor 1 (IGF-I) and glucose concentrations. The impact of environmental stressors was evaluated using plasma cortisol concentration. Heifers that had normal ovarian function had significantly higher IGF-I concentrations at commencement of the experiment (p = 0.008) and significantly higher plasma glucose concentrations at Day -2 (p = 0.040) and Day 4 (p = 0.043), than heifers with abnormal ovarian function. There was no difference between the mean pre-ovulatory cortisol concentrations of heifers that ovulated or did not ovulate. However, heifers that ovulated had higher cortisol concentrations at Day 4 (p = 0.056) and 6 (p = 0.026) after ovulation than heifers that did not ovulate.

Ovarian responses in Bos indicus heifers treated to synchronise ovulation with intravaginal progesterone releasing devices, oestradiol benzoate, prostaglandin F(2α) and equine chorionic gonadotrophin.

The objectives were: (i) improve understanding of the ovarian responses of Bos indicus heifers treated with different ovulation synchronisation protocols, (ii) compare ovarian responses of B. indicus heifers treated with intravaginal progesterone releasing device (IPRD)+oestradiol benzoate (ODB) versus a conventional prostaglandin F(2α) (PGF(2α)) protocol and (iii) investigate whether reducing the amount of progesterone (P(4)) in the IPRD, and treatment with equine chorionic gonadotrophin (eCG) would increase the proportion of heifers with normal ovarian function during the synchronised and return cycles. Two-year-old Brahman (n=30) and Brahman-cross (n=34) heifers were randomly allocated to three IPRD-treatment groups: (i) standard-dose IPRD (Cue-Mate(®) 1.56g P(4); n=17); (ii) half-dose IPRD (Cue-Mate(®) 0.78g P(4); n=15); (iii) half-dose IPRD+300IU eCG at IPRD removal (n=14), and a non-IPRD control group (iv) 2×PGF(2α) (500µg cloprostenol) on Days -16 and -2 (n=18). IPRD-treated heifers received 250µg cloprostenol at IPRD insertion (Day -10) and IPRD removal (Day -2) and 1mg ODB on Days -10 and -1. Ovarian function was evaluated by ultrasonography and plasma P(4) throughout the synchronised and return cycles. The mean diameter of the dominant follicle observed at 54-56h after IPRD removal, was greater for heifers which ovulated than heifers which did not ovulate (P<0.001; 14.5±1.1 vs. 9.3±0.6mm, respectively). The prevalence of IPRD-treated heifers with ovarian dysfunction (persistent CL, failure to re-ovulate, shortened luteal phase) was 39%. This relatively high prevalence of ovarian dysfunction may explain the commonly reported, lower than expected pregnancy rates to FTAI in B. indicus heifers treated to synchronise ovulation.

Pregnancy rates after fixed-time artificial insemination of Brahman heifers treated to synchronize ovulation with low-dose intravaginal progesterone releasing devices, with or without eCG.

The objective was to determine whether eCG in an ovulation synchronization protocol with an intravaginal progesterone (P(4))-releasing device (IPRD) containing a low dose of P(4) improves pregnancy rate (PR) to fixed-time AI (FTAI) in Bos indicus heifers. Day 0, 2 y old Brahman heifers were allocated to either eCG+ (n = 159) or eCG- (n = 157) treatment groups. All heifers were weighed, body condition scored (BCS), and ultrasonographically examined to measure uterine horn diameter and presence of a CL. On Day 0, all heifers received a low-dose IPRD (0.78 g P(4)) and 1 mg of estradiol benzoate (EB) im. On Day 8, the IPRD was removed, all heifers received 500 µg cloprostenol im, and those in the eCG+ treatment group received 300 IU of eCG im. On Day 9, all heifers received 1 mg EB im. All heifers were FTAI 52 to 56 h after IPRD removal. Ten days after FTAI, heifers were exposed to bulls. Heifers were diagnosed as pregnant to FTAI, natural mating, or not detectably pregnant (NDP) 65 d after FTAI. Treatment with eCG+ as compared to eCG- did not affect PR to FTAI (28.9 vs 30.6%; P = 0.590), natural mating (51.3 vs 47.7%; P = 0.595), or overall (65.4 vs 63.7%; P = 0.872). Mean live weight gain from Days 0 to 65 d post-FTAI was higher in heifers pregnant to FTAI (72.29 ± 4.26 kg; P = 0.033) and overall (66.83 ± 3.65 kg; P = 0.021), compared to heifers that were NDP (60.03 ± 3.16 kg). Uterine diameter group, 9-11, 12-13, and 14-20 mm (26.2, 31.3, and 33.3%; P = 0.256), presence and absence of CL (29.8 vs 29.4%; P = 0.975), AI technicians 1, 2, and 3 (32.6, 28.8, and 22.4%; P = 0.293) and sires A, B, and C (23.9, 36.0 and 27.0%; P = 0.122) had no effect on PR to FTAI, natural mating, or overall. In conclusion, treatment of primarily cycling Brahman heifers with 300 IU eCG in conjunction with a low P(4)-dose (0.78 g) IPRD and EB to synchronize ovulation, did not improve PR after FTAI, natural mating, or overall.

A single nucleotide polymorphism-derived regulatory gene network underlying puberty in 2 tropical breeds of beef cattle.

Harsh tropical environments impose serious challenges on poorly adapted species. In beef cattle, tropical adaptation in the form of temperature and disease resistance, coupled with acclimatization to seasonal and limited forage, comes at a cost to production efficiency. Prominent among these costs is delayed onset of puberty, a challenging phenotype to manipulate through traditional breeding mechanisms. Recently, system biology approaches, including gene networks, have been applied to the genetic dissection of complex phenotypes. We aimed at developing and studying gene networks underlying cattle puberty. Our starting material comprises the association results of ~50,000 SNP on 22 traits, including age at puberty, and 2 cattle breed populations: Brahman (n = 843) and Tropical Composite (n = 866). We defined age at puberty as the age at first corpus luteum (AGECL). By capturing the genes harboring mutations minimally associated (P < 0.05) to AGECL or to a set of traits related with AGECL, we derived a gene network for each breed separately and a third network for the combined data set. At the intersection of the 3 networks, we identified candidate genes and pathways that were common to both breeds. Resulting from these analyses, we identified an enrichment of genes involved in axon guidance, cell adhesion, ErbB signaling, and glutamate activity, pathways that are known to affect pulsatile release of GnRH, which is necessary for the onset of puberty. Furthermore, we employed network connectivity and centrality parameters along with a regulatory impact factor metric to identify the key transcription factors (TF) responsible for the molecular regulation of puberty. As a novel finding, we report 5 TF (HIVEP3, TOX, EYA1, NCOA2, and ZFHX4) located in the network intersecting both breeds and interacting with other TF, forming a regulatory network that harmonizes with the recent literature of puberty. Finally, we support our network predictions with evidence derived from gene expression in hypothalamic tissue of adult cows.

Relationship among seminal quality measures and field fertility of young dairy bulls using low-dose inseminations.

Optimal use of genetically superior bulls through artificial insemination (AI) is highly dependent on precise assessment of seminal quality which allows for reasonable estimations of field fertility with normal or low-dose inseminations. In the present study, seminal measures such as sperm motility and morphology, sperm viability, sperm DNA fragmentation, and the ability of the sperm to display an acrosome reaction were tested. The relationships between field fertility and the seminal measures were investigated using 3 ejaculates from each of 195 bulls (156 Holstein and 39 Jersey) participating in a progeny test program. A range of AI doses, varying from 2×10(6) to 15×10(6) sperm/straw, was obtained by a controlled dilution process applied to each ejaculate. The different AI doses were distributed at random among 75,610 experimental first inseminations in 4,721 herds and 208 AI technicians. Most of the seminal measures appeared to contain a predictive value for the nonreturn to estrus at 56 d post-AI (NRR56) regardless of the number of sperm per AI dose and can be regarded as noncompensable sperm traits. But, due to correlations between the individual measures, the best model for describing (and predicting) NRR56 was based on sperm concentration and viability in the neat (raw) semen, and post-thaw sperm viability. The statistical models for describing NRR56 included the following explanatory variables: strength of the estrus, number of sperm per AI dose, breed, parity, and random components representing herds and AI technicians. The present results show that the most precise estimation of a bull's NRR56 can be achieved through flow cytometric detection of sperm concentration and viability in neat semen as well as flow cytometric detection of post-thaw sperm viability.

Malignant Sertoli cell tumour in a young Simmenthal bull--clinical and pathological observations.

Tumours are rare in the bovine testicle. A case of malignant Sertoli cell tumour in a 29-month-old Simmenthal bull that was hospitalized with a history of severe unilateral scrotal swelling is reported. On inspection and palpation, the scrotal sac was found enlarged with fluctuant content in the right side. The right testicle was enlarged, hard and indolent. Also the right plexus pampiniformis and funiculus spermaticus were enlarged. Sonograms revealed severe changes in the right testicle with a loss of homogeneity and multiple hyperechogenic areas. After slaughter, the scrotum with testicles were removed and evaluated pathologically. On section, the right testicle contained areas of necrosis, haemorrhage, and mineralization. Histology showed Sertoli cells in tubular structures surrounded by dense fibrous stroma replacing normal testicular tissue. Both lymphatic and blood vessels were infiltrated by neoplastic cells. Immunohistochemically, the neoplastic cells stained positive for vimentin and negative for cytokeratin and S-100. Based on the pathological observations a diagnosis of right-sided malignant Sertoli cell tumour with vascular invasion and hydrocele was established.

Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility.

Extended semen doses from some boars used for AI have been shown to develop high levels of sperm DNA fragmentation during storage. Studies in other animals and humans have shown that if DNA damage is present in a certain percentage of the sperm cells the fertility potential of the semen sample is reduced. The objectives of the present study was to determine the relationship between sperm DNA fragmentation measured using the sperm chromatin structure assay (SCSA) in extended stored semen and field fertility in the boar. Three ejaculates from each of 145 boars were collected. Preparation of the semen doses included dilution with an EDTA extender and storage for up to 72 h post collection. The semen doses were assessed using flow cytometric methods for the percentage of viable sperm (PI/SYBR-14) and sperm DNA fragmentation (SCSA) at 0, 24, 48, and 72 h. A total of 3276 experimental inseminations in Danish breeding herds were conducted. The results showed that for 11 (7.6%) of the boars at least one of the three samples showed a value of DNA fragmentation index (DFI) above 20% within the storage period. Total number of piglets born (litter size) for Hampshire, Landrace and Danish Large White boars was, respectively, 0.5, 0.7 and 0.9 piglets smaller per litter when DFI values were above 2.1% as opposed to below this value. In conclusion the SCSA technique appears to be able to identify individuals with lower fertility with respect to litter size, and could in the future be implemented by the pig industry after a cost-benefit analysis.