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.     

In vitro evaluation of cryopreserved bovine sperm and its relation to field fertility in fixed‐time artificial insemination

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed‐time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H₂O₂ production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H₂O₂ production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H₂O₂ production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation‐like status of the acrosome may benefit probability of pregnancy in cows.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

Effects of exposure to artificial long days on milk yield,maternal insulin‐like growth factor 1 levels and kid growth rate in subtropical goats

This study was designed to determine whether any relationship exists between exposure to artificial long days, milk yield, maternal plasma insulin‐like growth factor 1 (IGF‐1) levels, and kid growth rate in goats. One group of lactating goats was maintained under naturally decreasing day length (control group; n = 19), while in another one, they were kept under artificial long days (LD group; n = 19). Milk yield was higher in goats from the LD group than that in the control group (P < 0.05). Maternal IGF‐1 levels at day 57 of lactation were higher (P < 0.05) in goats from the LD group than the levels in the control group and were positively correlated with the total milk yields per goat at days 43 and 57 of lactation (r = 0.77 and r = 0.84, respectively; P < 0.01). Daily weight gain at week 4 was higher (P < 0.01) in kids from the LD group than that in kids from the control group and was correlated with total and average IGF‐1 maternal levels (r = 0.60 and r = 0.60, P < 0.05). It was concluded that submitting lactating goats to artificial long days increases milk yield, plasma IGF‐1 maternal levels and the growth rate of the kids.     

The effect of fixed-time artificial insemination protocol initiated at different stages of the estrous cycle on follicle development and ovulation in gilts

Hormonal products have been developed for fixed-time artificial insemination (FTAI) to improve the efficiency of swine production. Here, we evaluated the effect of an FTAI protocol initiated during different phases of the estrous cycle on follicle development and ovulation in gilts. A total of 36 gilts were equally divided into three groups designated as the luteal (L), follicular (F), and post-ovulation (O) groups and fed with 20 mg of altrenogest for 18 days, followed by intramuscular injection of 1000 IU PMSG at 42 h after withdrawal of altrenogest, and 100 μg of GnRH after an 80-h interval. The L group had the highest number of follicles 4–6 mm in diameter, as well as corpora hemorrhagica. The mRNA expression of caspase-9 in the L group were significantly lower than those in the O and F groups (P < 0.05), while CYP11A1 and VEGF mRNA expression levels were significantly higher (P < 0.05). Moreover, FSHR mRNA levels were significantly higher in the O group than in the L, F, and control groups (P < 0.05). LHCGR and CYP19A1 mRNA levels were the highest in the F group (P < 0.05). Thus, the changes in the expression of genes associated with follicular development, maturation, and ovulation identified in this study indicated that initiation of the FTAI protocol during the luteal phase induced a better environment for follicle development and ovulation in gilts.     

Effect of body condition score and reuse of progesterone‐releasing intravaginal devices on conception rate following timed artificial insemination in Nelore cows

This study had the aim of investigating the efficiency of timed artificial insemination (TAI) through the progesterone‐releasing intravaginal device (PRID), used in new condition and for the second and third times in Nelore cows. The effects of device reuse and body condition score (BCS) on the conception rate (CR) were evaluated in 1,122 multiparous Nelore cows (mean BCS of 2.7 ± 0.4), which were randomly distributed into three groups that received new (n = 330), once (n = 439) and twice used (n = 353) PRID. Among the 1,122 females that underwent TAI, 573 became pregnant, thus representing an overall CR of 51.06%. Cows with BCS between 2.75 and 4.0 had greater (p < .0001) CR (69.75%) than cows with BSC of 2.0–2.5 (32.98%). It was observed that the CR through using PRID was 60.00%, 51.71% and 41.93% for new, once and twice used PRID, respectively, with difference between all groups (p < .0001). Under tropical conditions, animals with BCS greater than 2.5 had a higher CR, and the CR decreased proportionally with the number of times that the PRID had been used.     

Effect of ovsynch versus prostaglandin F2α protocol on estrus response,ovulation rate,timing of ovulation and pregnancy per artificial insemination in Sahiwal cows

Sahiwal cow is Bos indicus which is an important dairy breed of tropical and sub‐tropical region. Research on reproduction is rare in this breed. The objectives of the present study were to determine the effect of Ovsynch (OVS) versus prostaglandin F_2α (PG) protocol on estrus response and its intensity, ovulation rate, timing of ovulation and pregnancy per artificial insemination (AI) in Sahiwal cows. Experimental cows (n = 80) were of mixed parity, lactating, suckled, ≥120 days postpartum with body condition score 3.08 ± 0.34 and 375‐475 kg of body weight which were randomly assigned to receive either OVS (n = 46) or PG (n =34) protocol. Cows were inseminated twice at 12 and 24 h after second gonadotropin releasing hormone in the OVS group, and 72 and 84 h after administration of prostaglandin F_2α in PG group, respectively. The results revealed that estrus response did not differ (P > 0.05) and was 87% in OVS and 78% in PG cows. Ovulation rate did not differ (P > 0.05) and was 50% in both, OVS and PG cows. The pregnancy per AI did not differ (P > 0.05) and was 43% in OVS compared to 31% in PG cows. It is concluded that estrus response, ovulation rate and pregnancy per AI of OVS protocol is the same as PG in Sahiwal cows.     

A 57‐bp deletion in the ovine KAP6‐1 gene affects wool fibre diameter

High glycine–tyrosine keratin‐associated proteins (HGT‐KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction – single‐strand conformational polymorphism (PCR‐SSCP) analysis to screen for variation in a gene encoding the ovine HGT‐KAP6‐1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57‐bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6‐1 affects wool fibre diameter‐associated traits and that the 57‐bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.     

Genetic variation at the alpha‐1‐fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds

Escherichia coli F18 bacteria producing enterotoxins and/or shigatoxin (ETEC/STEC) are main pathogens that cause oedema disease and postweaning diarrhoea in piglets, and alpha‐1‐fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of ETEC F18 receptor. The genetic variations at nucleotide position 307 in open reading frame of FUT1 gene in one wild boar breed and 20 western commercial and Chinese native pig breeds were investigated by polymerase chain reaction–restriction fragment length polymorphism. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in western pig breeds and the Chinese Taihu (including Meishan pig, Fengjing pig and Erhualian pig), Huai and Lingao pig breeds; only Duroc and Pietrain possessed the resistant AA genotype, while the wild boar and other Chinese pig breeds only presented the susceptible genotype GG. The results indicated that Chinese native pig breeds lack genetic factors providing resistance to ETEC F18 bacteria. The resistant allele to ETEC F18 might originate from European wild boar. It was inferred that oedema and postweaning diarrhoea caused by ETEC F18 have close relationship with the growth rate, which can explain why on the contrary Chinese native pig breeds have stronger resistance to oedema and postweaning diarrhoea in piglets compared with western pig breeds.     

Rno‐miR‐425‐5p targets the DLST and SLC16A1 genes to reduce liver damage caused by excessive energy mobilization under cold stress

MicroRNAs (miRNAs) are a class of single‐stranded non‐coding small RNA molecules, which participate in the regulation of many physiological processes, and play a crucial role in cancer, metabolism and other processes. Rno‐miR‐425‐5p has been shown to play a role in the response to cold stress. To explore the mechanism by which rno‐miR‐425‐5p regulates the response to cold stress, we analysed the candidate target genes of rno‐miR‐425‐5p. After verification in rat hepatocyte BRL cells and in rat liver tissue, we identified several target genes that were altered in expression in response to cold stress. In rat liver tissue, the expression of rno‐miR‐425‐5p was significantly increased and the expression levels of target genes DLST and SLC16A1 were decreased under cold stress. The miRNA and mRNA levels were analysed by quantitative real‐time PCR and the protein levels were detected by Western blot analysis. Combined with the results of bioinformatic analysis, we concluded that rno‐miR‐425‐5p reduced the expression of DLST and SLC16A1, inhibiting energy release from the tricarboxylic acid cycle and preventing the liver from being injured by excessive energy mobilization.     

Selenium‐enriched Saccharomyces cerevisiae improves growth,antioxidant status and selenoprotein gene expression in Arbor Acres broilers

One hundred and fifty 7‐day‐old Arbor Acres broilers were randomly assigned into five groups: group 1 served as a control that was fed a basal diet without selenium (Se) supplementation; groups 2, 3 and 4 were fed the basal diet supplemented with 0.15, 0.5 and 1.5 mg Se as Se‐enriched Saccharomyces cerevisiae (SSC) per kg of diet; and group 5 was fed the basal diet supplemented with 0.15 mg per kg of Se as sodium selenite (SS). Growth performance, glutathione peroxidase (GP_X) and superoxide dismutase (SOD) activities, total antioxidant capacity (T‐AOC), and malondialdehyde (MDA) content in plasma and liver, and cellular glutathione peroxidase (GP_X‐1) and phospholipid hydroperoxide glutathione peroxidase (GP_X‐4) mRNA levels in liver were determined. Compared with group 1, groups 2–4 exhibited higher body weights (p < 0.05), lower feed/gain ratios, and higher GP_X activities in plasma (p < 0.05) and GP_X and SOD activities and GP_X‐1 and GP_X‐4 mRNA levels in liver (p < 0.05). Compared with group 5, group 2 exhibited higher GP_X activity in plasma on day 21 (p < 0.05). Compared with group 2 and 5, group 3 exhibited lower MDA content in plasma on day 7 (p < 0.05), higher GP_X activity in plasma, SOD activity and GP_X‐1 mRNA levels in liver on day 14 and 21 (p < 0.05), and higher GP_X‐4 mRNA levels on day 14 (p < 0.05). Compared with group 4, group 3 exhibited lower MDA contents in plasma on day 14 (p < 0.05) and in liver on day 21 (p < 0.05), higher T‐AOC in plasma and higher GP_X‐1 mRNA levels on day 14 and 21 (p < 0.05), and higher SOD activity in plasma and higher SOD and GP_X activities in liver on day 21 (p < 0.05). Thus, SSC improves growth and antioxidant status of broilers; the short‐term bioavailability of SS was faster than that of SSC, but the long‐term bioavailability of SSC was greater than SS.     

Polo‐like kinase 1 inhibition results in misaligned chromosomes and aberrant spindles in porcine oocytes during the first meiotic division

Polo‐like kinase 1 (Plk1), a type of serine/threonine protein kinase, has been implicated in various functions in the regulation of mitotic processes. However, these kinase's roles in meiotic division are not fully understood, particularly in the meiotic maturation of porcine oocytes. In this study, the expression and spatiotemporal localization of Plk1 were initially assessed in the meiotic process of pig oocytes by utilizing Western blotting with immunofluorescent staining combined with confocal microscopy imaging technique. The results showed that Plk1 was expressed and exhibited a dynamic subcellular localization throughout the meiotic process. After germinal vesicle breakdown (GVBD), Plk1 was detected prominently around the condensed chromosomes and subsequently exhibited a similar subcellular localization to α‐tubulin throughout subsequent meiotic phases, with particular enrichment being observed near spindle poles at MI and MII. Inhibition of Plk1 via a highly selective inhibitor, GSK461364, led to the failure of first polar body extrusion in porcine oocytes, with the majority of the treated oocytes being arrested in GVBD. Further subcellular structure examination results indicated that Plk1 inhibition caused the great majority of oocytes with spindle abnormalities and chromosome misalignment during the first meiotic division. The results of this study illustrate that Plk1 is critical for the first meiotic division in porcine oocytes through its influence on spindle organization and chromosome alignment, which further affects the ensuing meiotic cell cycle progression.     

Methyl donors dietary supplementation to gestating sows diet improves the growth rate of offspring and is associating with changes in expression and DNA methylation of insulin‐like growth factor‐1 gene

The study aimed to investigate the effects of maternal dietary methyl donors on the performance of sows and their offspring, and the associated hepatic insulin‐like growth factor‐1 (IGF‐1) expression of the offspring. A total of 24 multiparous sows were randomly fed the control (CON) or the CON diet supplemented with methyl donors (MD) at 3 g/kg betaine, 15 mg/kg folic acid, 400 mg/kg choline and 150 μg/kg VB₁₂, from mating until delivery. After farrowing, sows were fed a common lactation diet through a 28‐days lactation period and six litters per treatment were selected to be fed until at approximately 110 kg BW. Maternal MD supplementation resulted in greater birthweight (p < 0.05) and increased the piglet weights (p < 0.01) and litter weights (p < 0.05) at the age of day 28, compared with that in CON group. The offspring pigs in the MD group had greater ADG (p < 0.05) and tended to lower F:G ratio (p = 0.07) compared with that of CON group from day 28 to 180 of age. The offspring pigs from MD group had greater serum IGF‐1 concentrations and expressions of hepatic IGF‐1 gene and muscular IGF‐1 receptor (IGF‐1r) protein at birth (p < 0.05), and greater hepatic IGF‐1 protein (p = 0.03) and muscular IGF‐1r gene expressions (p < 0.05) at slaughter, than that from the CON group. Moreover, the methylation at the promoter of IGF‐1 gene in the liver of newborn piglets and finishing pigs was greater in the MD group than that of the CON group (p < 0.05). In conclusion, maternal MD supplementation throughout gestation could enhance the birthweight and postnatal growth rate of offspring, associated with an increased expression of the IGF‐1 gene and IGF‐1r, as well as the altered DNA methylation of IGF‐1 gene promotor.