Low Fertility Groups Research Articles

Identification of structural variation related to spawn capability of Penaeus vannamei

BackgroundThe genetic basis underlying spawning abilities in the Pacific white shrimp, Penaeus vannamei, remains largely unexplored. To investigate genetic variations potentially related to reproductive performance, a systematic bioinformatic analysis was conducted to identify structural variations (SVs) with different polymorphic spectra in P. vannamei with high fertility (HF) and low fertility (LF).ResultsA total of 2,323 and 1,859 SV events were identified exclusively in the HF and LF groups, respectively. These SVs were mapped to 277 genes in the HF group and 231 genes in the LF group. Gene Ontology (GO) enrichment analysis based on SNPs (single nucleotide polymorphism) and SVs revealed several neural-related processes, suggesting the importance of neural regulation in reproduction. Notably, we identified a set of promising genes, including Cttn, Spast, Ppp4c, Spire1, Lhcgr, and Ftz-f1, which may enhance fertility in shrimp.ConclusionIn conclusion, this study is the first to establish a link between SVs and reproductive traits in P. vannamei. The promising genes discovered have the potential to serve as crucial markers for enhancing reproductive traits through targeted genotyping.

Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0–5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0–5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (−0.0002 ± 0.07 vs. −0.266 ± 0.09), kurharm0 (−0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0–5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. −0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

Ovarian function and endocrine phenotypes of lactating dairy cows during the estrous cycle are associated with genomic-enhanced predictions of fertility potential

The objectives of this prospective cohort study were to characterize associations among genomic merit for fertility with ovarian and endocrine function and the estrous behavior of dairy cows during an entire nonhormonally manipulated estrous cycle. Lactating Holstein cows entering their first (n = 82) or second (n = 37) lactation had ear-notch tissue samples collected for genotyping using a commercial genomic test. Based on genomic predicted transmitting ability values for daughter pregnancy rate (gDPR), cows were classified into high (Hi-Fert; gDPR > 0.6, n = 36), medium (Med-Fert; gDPR -1.3 to 0.6, n = 45), and low fertility (Lo-Fert; gDPR < -1.3, n = 38) groups. At 33 to 39 DIM, cohorts of cows were enrolled in the Presynch-Ovsynch protocol for synchronization of ovulation and initiation of a new estrous cycle. Thereafter, the ovarian function and endocrine dynamics were monitored daily until the next ovulation by transrectal ultrasonography and concentrations of progesterone (P4), estradiol, and FSH. Estrous behavior was monitored with an ear-attached automated estrus detection system that recorded physical activity and rumination time. Overall, we observed an association between fertility group and the ovarian and hormonal phenotype of dairy cows during the estrous cycle. Cows in the Hi-Fert group had greater circulating concentrations of P4 than cows in the Lo-Fert group from d 4 to 13 after induction of ovulation and from day -3 to -1 before the onset of luteolysis. The frequency of atypical estrous cycles was 3-fold greater for cows in the Lo-Fert than the Hi-Fert group. We also observed other modest associations between genomic merit for fertility with the follicular dynamics and estrous behavior. We found several associations between milk yield and parity with ovarian, endocrine, and estrous behavior phenotypes as cows with greater milk yield and in the second lactation were more likely to have unfavorable phenotypes. These results demonstrate that differences in reproductive performance between cows of different genomic merit for fertility classified based on gDPR may be partially associated with circulating concentrations of P4, the incidence of atypical phenotypes during the estrous cycles, and, to a lesser extent, the follicular wave dynamics. The observed physiological and endocrine phenotypes might help explain part of the differences in reproductive performance between cows of superior and inferior genomic merit for fertility.

Construction of a circRNA- lincRNA-lncRNA-miRNA-mRNA ceRNA regulatory network identifies genes and pathways linked to goat fertility.

Background: There is growing interest in the genetic improvement of fertility traits in female goats. With high-throughput genotyping, single-cell RNA sequencing (scRNA-seq) is a powerful tool for measuring gene expression profiles. The primary objective was to investigate comparative transcriptome profiling of granulosa cells (GCs) of high- and low-fertility goats, using scRNA-seq. Methods: Thirty samples from Ji'ning Gray goats (n = 15 for high fertility and n = 15 for low fertility) were retrieved from publicly available scRNA-seq data. Functional enrichment analysis and a literature mining approach were applied to explore modules and hub genes related to fertility. Then, interactions between types of RNAs identified were predicted, and the ceRNA regulatory network was constructed by integrating these interactions with other gene regulatory networks (GRNs). Results and discussion: Comparative transcriptomics-related analyses identified 150 differentially expressed genes (DEGs) between high- and low-fertility groups, based on the fold change (≥5 and ≤-5) and false discovery rate (FDR <0.05). Among these genes, 80 were upregulated and 70 were downregulated. In addition, 81 mRNAs, 58 circRNAs, 8 lincRNAs, 19 lncRNAs, and 55 miRNAs were identified by literature mining. Furthermore, we identified 18 hub genes (SMAD1, SMAD2, SMAD3, SMAD4, TIMP1, ERBB2, BMP15, TGFB1, MAPK3, CTNNB1, BMPR2, AMHR2, TGFBR2, BMP4, ESR1, BMPR1B, AR, and TGFB2) involved in goat fertility. Identified biological networks and modules were mainly associated with ovary signature pathways. In addition, KEGG enrichment analysis identified regulating pluripotency of stem cells, cytokine-cytokine receptor interactions, ovarian steroidogenesis, oocyte meiosis, progesterone-mediated oocyte maturation, parathyroid and growth hormone synthesis, cortisol synthesis and secretion, and signaling pathways for prolactin, TGF-beta, Hippo, MAPK, PI3K-Akt, and FoxO. Functional annotation of identified DEGs implicated important biological pathways. These findings provided insights into the genetic basis of fertility in female goats and are an impetus to elucidate molecular ceRNA regulatory networks and functions of DEGs underlying ovarian follicular development.

Effect of reproductive management programs that prioritized artificial insemination at detected estrus or timed artificial insemination on the economic performance of primiparous Holstein cows of different genetic merit for fertility

The objective of this randomized controlled experiment was to evaluate the effect of reproductive management programs that prioritized artificial insemination (AI) at detected estrus (AIE) or timed AI (TAI) during the first lactation on the economic performance of dairy cows of different genomically enhanced predicted transmitting ability for fertility. Lactating primiparous Holstein cows from 6 commercial farms were stratified into high, medium, and low fertility groups based on a reproduction index value calculated from multiple genomically enhanced predicted transmitting abilities to predict the number of days to achieve pregnancy. Within herd and fertility group, cows were randomly assigned either to a program that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows not AIE for all AI services or another that prioritized TAI and had an extended voluntary waiting period for first service and prioritized TAI for second and greater AI services (P-TAI; n = 1,338). Cash flow (CF) per cow accumulated for the experimental (first) and second calving interval (CIN) and cash flow per slot per 28 mo after calving in the experimental lactation were calculated. Market and rearing heifer cost values were used for estimating CF. For cows in the high fertility group, a positive effect of delayed pregnancy on milk income during the first lactation was observed (+$248 for P-TAI) but was insufficient to generate significant differences in CF between treatments mainly because of milk income compensation in the second lactation (+$125 for P-AIE) and minor reductions in reproductive cost and gains in calf value for the P-AIE treatment. In this regard, CF for 2 CIN was greater for the P-TAI treatment by $61 and $86 for market and rearing replacement heifer cost, respectively. Similarly, CF per slot was favorable to the P-TAI treatment but only by $13 and $47 for market and rearing replacement heifer cost, respectively. For cows in the low fertility group, CF was numerically in favor of the P-AIE treatment due to a pregnancy and herd exit dynamics that resulted in gains in milk income over feed cost during the first ($29) and second ($113) lactation. Differences in CF for the 2 CIN were $58 and $47 for market or rearing heifer value, respectively, and $77 and $19 for market and rearing heifer values, respectively for the slot analysis. Differences in CF between cows of different genetic merit for fertility were consistent across treatment and estimation method. Of note, cows in the low fertility group had greater CF than cows in the high fertility group in all comparisons, ranging from $198 per cow for 2 CIN to as much as $427 per slot. For the low fertility group, greater milk production contributed directly (milk income over feed cost) and indirectly (reduced culling) to increased CF. We concluded that genetic merit for fertility and CF are associated because cows of inferior genetic potential for fertility had greater CF than cows of superior genetic for fertility despite some increased costs and reduced revenues. Also, the magnitude of the CF differences observed for cows of different genetic merit for fertility managed with the P-AIE or P-TAI program may be valuable to commercial dairy farms but did not allow to conclusively support the choice of a type of reproductive management strategy for cows of different genetic merit for fertility.

Effect of reproductive management programs that prioritized artificial insemination at detected estrus or timed artificial insemination on the reproductive performance of primiparous Holstein cows of different genetic merit for fertility

Our objective was to compare reproductive outcomes of primiparous lactating Holstein cows of different genetic merit for fertility submitted for insemination with management programs that prioritized artificial insemination (AI) at detected estrus (AIE) or timed AI (TAI). Moreover, we aimed to determine whether subgroups of cows with different fertility potential would present a distinct response to the reproductive management strategies compared. Lactating primiparous Holstein cows (n = 6 commercial farms) were stratified into high (Hi-Fert), medium (Med-Fert), and low (Lo-Fert) genetic fertility groups (FG) based on a Reproduction Index value calculated from multiple genomic-enhanced predicted transmitting abilities. Within herd and FG, cows were randomly assigned either to a program that prioritized TAI and had an extended voluntary waiting period (P-TAI; n = 1,338) or another that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows, not AIE. Cows in P-TAI received first service by TAI at 84 ± 3 d in milk (DIM) after a Double-Ovsynch protocol, were AIE if detected in estrus after a previous AI, and received TAI after an Ovsynch-56 protocol at 35 ± 3 d after a previous AI if a corpus luteum (CL) was visualized at nonpregnancy diagnosis (NPD) 32 ± 3 d after AI. Cows with no CL visualized at NPD received TAI at 42 ± 3 d after AI after an Ovsynch-56 protocol with progesterone supplementation (P4-Ovsynch). Cows in P-AIE were eligible for AIE after a PGF2α treatment at 53 ± 3 DIM and after a previous AI. Cows not AIE by 74 ± 3 DIM or by NPD 32 ± 3 d after AI received P4-Ovsynch for TAI at 74 ± 3 DIM or 42 ± 3 d after AI. Binary data were analyzed with logistic regression, count data with Poisson regression, continuous data by ANOVA, and time to event data by Cox's proportional hazard regression. Pregnancy per AI (P/AI) to first service was greater for cows in the Hi-Fert (59.8%) than the Med-Fert (53.6%) and Lo-Fert (47.7%) groups, and for the P-TAI (58.7%) than the P-AIE (48.7%) treatment. Overall, P/AI for all second and subsequent AI combined did not differ by treatment (P-TAI = 45.2%; P-AIE = 44.5%) or FG (Hi-Fert = 46.1%; Med-Fert = 46.0%; Lo-Fert = 42.4%). The hazard of pregnancy after calving was greater for the P-AIE than the P-TAI treatment [hazard ratio (HR) = 1.27, 95% CI: 1.17 to 1.37)], and for the Hi-Fert than the Med-Fert (HR = 1.16, 95% CI: 1.05 to 1.28) and Lo-Fert (HR = 1.34, 95% CI: 1.20 to 1.49) groups. More cows in the Hi-Fert (91.2%) than the Med-Fert (88.4%) and Lo-Fert (85.8%) groups were pregnant at 200 DIM. Within FG, the hazard of pregnancy was greater for the P-AIE than the P-TAI treatment for the Hi-Fert (HR = 1.41, 95% CI: 1.22 to 1.64) and Med-Fert (HR = 1.28, 95% CI: 1.12 to 1.46) groups but not for the Lo-Fert group (HR = 1.13, 95% CI: 0.98 to 1.31). We conclude that primiparous Holstein cows of superior genetic merit for fertility had better reproductive performance than cows of inferior genetic merit for fertility, regardless of the type of reproductive management used. In addition, the effect of programs that prioritized AIE or TAI on reproductive performance for cows of superior or inferior genetic merit for fertility depended on the outcomes evaluated. Thus, programs that prioritize AIE or TAI could be used to affect certain outcomes of reproductive performance or management.

Identification of runs of homozygosity associated with male fertility in Italian Brown Swiss cattle.

Intensive selection for improved productivity has been accompanied by an increase in inbreeding rates and a reduction in genetic diversity. The increase in inbreeding tends to impact performance, especially fitness-related traits such as male fertility. Inbreeding can be monitored using runs of homozygosity (ROH), defined as contiguous lengths of homozygous genotypes observed in an individual's chromosome. The goal of this study was to evaluate the presence of ROH in Italian Brown Swiss cattle and assess its association with bull fertility. First, we evaluated the association between ROH and male fertility using 1,102 Italian Brown Swiss bulls with sire conception rate records and 572K SNPs spanning the entire genome. Second, we split the entire population into 100 high-fertility and 100 low-fertility bulls to investigate the potential enrichment of ROH segments in the low-fertility group. Finally, we mapped the significant ROH regions to the bovine genome to identify candidate genes associated with sperm biology and male fertility. Notably, there was a negative association between bull fertility and the amount of homozygosity. Four different ROH regions located in chromosomes 6, 10, 11, and 24 were significantly overrepresented in low-fertility bulls (Fisher's exact test, p-value <0.01). Remarkably, these four genomic regions harbor many genes such as WDR19, RPL9, LIAS, UBE2K, DPF3, 5S-rRNA, 7SK, U6, and WDR7 that are related to sperm biology and male fertility. Overall, our findings suggest that inbreeding and increased homozygosity have a negative impact on male fertility in Italian Brown Swiss cattle. The quantification of ROH can contribute to minimizing the inbreeding rate and avoid its negative effect on fitness-related traits, such as male fertility.

Effect of Dhatryadi Ghrithm in “Diminishing Ovarian Reserve”

Background: Diminishing ovarian reserve (DOR) describes women of reproductive age with regular cycles, mostly ovulatory, whose response to ovarian stimulation is reduced compared to women of comparable age. The most appropriate correlation of DOR can be done with Dhatukshaya Vandya. Dhatraydi Ghrithm mentioned in Sahasra Yogam is said to have implicit effect in enhancing reproductive potential. &#x0D; Objectives: To evaluate the effect of Dhatryadi Ghrithm in “Diminishing ovarian reserve”.&#x0D; Materials and methods: It was a pre and post interventional study done during the time period July 2017 to December 2018 with a sample size of 20. Females between the age group of 20-35 years, diagnosed with DOR attending Outpatient Department of Govt Ayurveda College Hospital for Women and Children, Poojappura, Thiruvananathapuram were selected. Serum Anti Mullerian Hormone (AMH), Serum Estradiol, Antral Follicular Count (AFC) and LH/FSH ratio, Ovarian volume was assessed on the 2nd day of the cycle after completing 90 days of Ghritha administration. Paired t test was used to analysis the data statistically. &#x0D; Results: 55 % of total study population were in the low fertility group and 45% confined to the very low fertility group.7 out of 20 patients obtained satisfactory level of fertility after 3 months of treatment. There was a considerable increase in mean value of AMH before and after treatment (2.62 to 9.01), p-value 0.001. AFC was also significantly increased after three months of treatment, p-value &lt;0.002. 3 patients were conceived during the study period.&#x0D; Conclusion: Study concluded that the Dhatryadi Ghrithm was found to be effective in increasing AMH and AFC, but it did not have any effect on serum Estradiol, LH/FSH ratio and in Ovarian volume.

Transcriptome analysis reveals mRNAs and long non-coding RNAs associated with fecundity in the hypothalamus of high-and low-fecundity goat.

As an important organ that coordinates the neuroendocrine system, the hypothalamus synthesizes and secretes reproductive hormones that act on the goat organism, thereby precisely regulating follicular development and reproductive processes in goats. However, it is still elusive to explore the mechanism of hypothalamic effects on goat fertility alone. Therefore, RNA-seq was used to analyze the gene expression in hypothalamic tissues of goats in high fertility group (HFG: litter size per litter ≥2) and low fertility group (LFG: litter size per litter = 1), and identified the differential lncRNAs and mRNAs and their associated pathways related to their fertility. The results showed that a total of 23 lncRNAs and 57 mRNAs were differentially expressed in the hypothalamic tissue of high and low fertility goats. GO terms and KEGG functional annotation suggest that DE lncRNAs and DE mRNAs were significantly enriched in hormone-related pathways regulating ovarian development, hormone synthesis and secretion, regulation of reproductive processes, Estrogen signaling pathway, Oxytocin signaling pathway and GnRH signaling pathway. And we constructed a co-expression network of lncRNAs and target genes, and identified reproduction-related genes such as NMUR2, FEZF1, and WT1. The sequencing results of the hypothalamic transcriptome have broadened our understanding of lncRNA and mRNA in goat hypothalamic tissue and provided some new insights into the molecular mechanisms of follicle development and regulation of its fertility in goats.

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high- and low-fertility bulls†.

Despite stringent quality control checks, some bulls with apparently normal semen quality yield lower than expected pregnancy rates. This study profiled the transcriptome and performed histological analysis of the bovine uterus in response to sperm from high-fertility (HF) and low-fertility (LF) bulls. Postmortem uterine biopsies and uterine explants were collected from heifers 12h after a fixed time artificial insemination (AI) to a synchronized estrus with frozen-thawed semen from five HF (fertility rate 4.01% ± 0.25) and five LF (fertility rate - 11.29% ± 1.11; mean ± SEM) bulls. Uterine biopsies were also collected from control (CTRL) heifers, which were not inseminated. RNA-sequencing and histological analysis were performed for differential gene expression and neutrophil quantification. In the HF treatment relative to CTRL heifers, there were 376 genes significantly differentially expressed in the endometrium with just one gene differentially expressed in the LF treatment relative to CTRL heifers. Comparing the HF and LF treatments directly, there were 40 significantly differentially expressed genes (P < 0.05). Transcriptomic analysis shows a predominant role for the inflammatory marker Interleukin-1 alpha, which was further confirmed by immunohistochemistry. Quantification of neutrophils in the endometrium showed a significant effect of sperm; however, there was no difference in neutrophil numbers between HF and LF groups. In conclusion, this novel study clearly shows a distinct inflammatory response to sperm in the endometrium and a divergent transcriptomic response to semen from HF and LF bulls.

Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos)

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.

Sperm Aldose Reductase B1 is not a predictor of bull in vivo fertility outcomes

Uncovering fertility biomarkers has become a major challenge for the dairy industry, where the fertility decline leads to important economic losses. In swine, seminal aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme, positively influenced fertility outcomes, probably through modulation of sperm fertilizing competence. In cattle, this protein has also been reported to be involved in epididymal sperm maturation. Against this background, the present work aimed to evaluate the potential relationship between the levels of AKR1B1 in bovine sperm and in vivo fertility outcomes. Thus, a total of 15 bulls were used to inseminate 577 heifers; in vivo fertility outcomes were calculated as 90-day non-return rates (NRR). In parallel, three ejaculates per bull were pooled and centrifuged for sperm AKR1B1 analysis with immunoblotting assay. Considering that a specific two-band pattern at 36 kDa and ~80 kDa was observed, two different ratios were calculated: 36 kDa form/total AKR1B1 and ~80 kDa form/total AKR1B1 (the total amount of AKR1B1 corresponded to the sum of the intensities of the two bands). There was no correlation(P > 0.05) between the levels of sperm AKR1B1 (36 kDa/total and /~80 kDa/total ratios) and NRR). Therefore, the 15 bulls were divided into high and low fertility groups using the NRR median value (high fertility group, 41.61 ± 1.19; and low fertility group, 36.22 ± 1.58), and the two AKR1B1 ratio levels were compared between those groups. There were no differences between NRR groups (P > 0.05) in the levels of sperm AKR1B1. These findings suggested that AKR1B1 may not be a good in vivo fertility predictor in bovine in terms of NRR. Yet, it cannot be discarded of the potential for an effect of this protein on other fertility indicators such as oocyte fertilisation or even embryo development.

Composition of Fatty Acids and Localization of SREBP1 and ELOVL2 Genes in Cauda Epididymides of Hu Sheep with Different Fertility.

The epididymis is an organ that transports, matures and stores sperm, and has functions such as secretion and absorption. Polyunsaturated fatty acid (PUFA) compositions in sperm membrane were changed during the process of epididymis maturation and influence the male fertility. This study aimed to investigate differences in crude fat and fatty acid content in cauda epididymis between high and low fertility of Hu sheep. One hundred and seventy-nine Hu ram lambs were fed from 56 days to 6 months under the same environment. After the feeding trial, all rams were slaughtered, and the body weight, testicular weight, epididymal weight and sperm density were measured. Pearson correlation analysis showed significantly moderate positive correlation between epididymal weight and sperm density and testicular weight. Eighteen rams were selected and divided into the high fertility group (H, n = 9) and low fertility group (L, n = 9) according to the epididymal weight, sperm density and histomorphology. The crude fat content, fatty acid profile and genes related to fatty acid metabolism were detected. The crude fat content, total fatty acid, total n-3 PUFA and docosahexaenoic acid (C22:6n-3, DHA) content of cauda epididymis in high fertility group was significantly higher than those in low fertility group (p < 0.05). However, the ratio of n-6/n-3 PUFA was significantly lower than that in group L (p < 0.05). Immunohistochemistry results showed that SREBP1 and ELOVL2 were expressed in pseudostratified columnar ciliated epithelium and smooth muscle cells. The mRNA expression of SREBP1 (p = 0.09) and ELOVL2 (p < 0.05) in the high fertility group were increased. In conclusion, the high expression of SREBP1 and ELOVL2 may contribute to high n-3 PUFA content in cauda epididymis of high-fertility Hu sheep.

Use of auxiliary techniques for assessing the reproductive capacity of natural service Nellore bulls

The aim of this study was to investigate whether parameters obtained by computer-assisted sperm analysis (CASA), infrared thermography (IRT), and testicular B-mode and Doppler ultrasound can be used as indicators of fertility in natural service Nellore bulls. Twenty-nine Nellore bulls were evaluated on days 66 (-D66), 38 (-D38), and 10 (-D10) before the beginning of natural service mating. In all assessments, semen samples were collected and sperm kinetics were evaluated by CASA, thermographic imaging of the scrotal and ocular region, testicular B-mode ultrasound, and Doppler ultrasound of the spermatic cord. At the end of natural service, the bulls were classified based on the pregnancy rate of individual batches into low fertility (pregnancy rate 66.57 ± 0.62 %), medium fertility (76.47 ± 0.51 %), and high fertility (84.80 ± 0.60 %) groups. No difference in the IRT parameters was observed between groups. High fertility animals exhibited higher average path velocity (VAP = 110.98 µm/s), straight line velocity (VSL = 87.05 µm/s), curvilinear velocity (VCL = 190.78 µm/s), pulsatility index (PI = 0.93), and resistive index (RI = 0.57) than low fertility animals (VAP = 100.02 µm/s; VSL = 79.84 µm/s; VCL = 173.22 µm/s; PI = 0.69; RI = 0.48). Positive correlations were observed between pregnancy rate and VSL (0.21), PI (0.28) and RI (0.32). In conclusion, IRT does not provide fertility indicators in Nellore bulls. The VAP, VSL and VCL obtained by CASA and PI and RI obtained by Doppler ultrasound can be used as indicators of fertility in Nellore bulls.

Establishment of a male fertility prediction model with sperm RNA markers in pigs as a translational animal model

BackgroundMale infertility is an important issue that causes low production in the animal industry. To solve the male fertility crisis in the animal industry, the prediction of sperm quality is the most important step. Sperm RNA is the potential marker for male fertility prediction. We hypothesized that the expression of functional genes related to fertilization will be the best target for male fertility prediction markers. To investigate optimum male fertility prediction marker, we compared target genes expression level and a wide range of field data acquired from artificial insemination of boar semen.ResultsAmong the genes related to acrosomal vesicle exocytosis and sperm–oocyte fusion, equatorin (EQTN), zona pellucida sperm-binding protein 4 (ZP4), and sperm acrosome membrane-associated protein 3 exhibited high accuracy (70%, 90%, and 70%, respectively) as markers to evaluate male fertility. Combinations of EQTN-ZP4, ZP4-protein unc-13 homolog B, and ZP4-regulating synaptic membrane exocytosis protein 1 (RIMS1) showed the highest prediction value, and all these markers are involved in the acrosome reaction.ConclusionThe EQTN-ZP4 model was efficient in clustering the high-fertility group and may be useful for selection of animal that has superior fertility in the livestock industry. Compared to the EQTN-ZP4 model, the ZP4-RIMS1 model was more efficient in clustering the low-fertility group and may be useful in the diagnosis of male infertility in humans and other animals. The appointed translational animal model and established biomarker combination can be widely used in various scientific fields such as biomedical science.

O-135 Sperm-borne small ribonucleic acid profile significantly impacts embryo development

Abstract Study question Do normozoospermic males with reduced pre-implantation embryo development have aberrant sperm small RNA profiles? Summary answer Small RNA sequencing suggests the small RNA profile may differ in normozoospermic males with low blastocyst development rates, compared to males with higher blastocyst progression. What is known already Current male factor infertility diagnostics are insufficient, with 30-50% of subfertile males having unknown etiology. Spermatozoa contain a complex, epigenetically-marked genome and a collection of RNAs and proteins, which are not adequately assessed by current diagnostic methods. The sperm small RNA payload is reportedly modified during epididymal transit and in response to paternal exposures, influencing which sperm small RNA species are delivered to the oocyte. Mechanistic animal studies and correlative human and animal studies have suggested that sperm small RNAs may be important for early embryonic development and health of offspring, though their diagnostic and therapeutic value are still unclear. Study design, size, duration Human semen samples were collected between April 2017 and August 2020 from a total of 56 male patients presenting to CReATe Fertility Centre for fertility evaluation. Clinical data was accessed retrospectively. All patients were normozoospermic, according to standard semen analysis and were using donor oocytes. Samples were divided into high (n = 20), average (n = 16), and low (n = 20) fertility groups based on their deviation (1 standard deviation) from the mean blastocyst rate. Participants/materials, setting, methods Semen analysis was undertaken immediately following sample collection and spermatozoa were isolated by centrifugation. Sperm small RNA was purified and eluted using the RNeasy and MiRNeasy Kits (Qiagen). Barcoded and amplified cDNA libraries were prepared from small RNA using the NEXTFLEX Small RNA-Seq Kit v3 (Bioo Scientific). Resulting libraries were pooled, size-selected to a range of 140-190 base pairs, denatured and diluted for sequencing. Single-end, 75 bp sequencing was performed using the NextSeq 550 (Illumina). Main results and the role of chance Sequencing generated approximately 300 million raw reads, with 30 samples exceeding 2 million reads included in the differential expression analysis. Most reads were mapped to rRNAs (69%), miRNAs (11%), and piRNAs (12%). However, transfer RNA fragments from tRNA-Gly-GCC and tRNA-Val-CAC were the most abundant sequences. Top annotated miRNAs include: miR-12136-5p; miR-21-5p; and miR-122-5p. Principal component analysis revealed 222 genes that were differentially expressed between the high (n = 14) and low (n = 11) fertility groups (p &amp;lt; 0.05). Interestingly, the top 50 differentially expressed sRNAs are sufficient to effectively cluster sperm with poor blastocyst development rates. Limitations, reasons for caution The results are limited by a relatively low sequencing depth (mean of 4.1 million reads per sample) and sample size. Fertility groups were determined by blastocyst rates, which can be confounded by non-sperm-derived variables, including technical skill, embryo culturing conditions, and maternal factors (though donor oocytes were used). Wider implications of the findings With additional validation, a clinically-useful panel of differentially expressed sperm small RNAs could be used to predict IVF success and evaluate therapies aimed at improving male reproductive health. Augmenting traditional semen analytics with diagnostic sperm small RNA analysis could reduce time to pregnancy and the psychosocial impacts of fertility treatment. Trial registration number Not applicable

Direct genomic value daughter pregnancy rate and services per conception are associated with characteristics of day-16 conceptuses and hormone signaling for maternal recognition of pregnancy in lactating Holstein cows

Our objective was to test the hypothesis that sorting Holstein dairy cows with high direct genomic value for daughter pregnancy rate (DGV-DPR) and low services per conception records improves interferon tau production and endometrial signaling for maternal recognition of pregnancy. Lactating Holstein cows (n = 86) were sorted by DGV-DPR into high- and low-fertility groups with the Clarifide ( https://www.zoetisus.com/animal-genetics/dairy/clarifide/clarifide.aspx# ) DNA test and low and high services per conception from farm records. High-fertility nonpregnant (NP) cows were not inseminated (n = 7) and served as controls. High-fertility pregnant (HP; n = 7) and low-fertility pregnant (LP; n = 6) cows received timed AI. Conceptuses, uterine flushings, endometrial biopsies, and peripheral blood mononuclear cells were collected on d 16 of pregnancy or the estrous cycle. Conceptuses from HP cows were longer ( P < 0.05) than those from LP conceptuses. Concentrations of interferon tau in uterine flushings were greater ( P < 0.05) in HP compared with LP and NP cows, positively correlated with DGV-DPR (r = +0.68; P < 0.05), and negatively correlated with services per conception (r = −0.59; P < 0.05). Endometrial ISG15 mRNA and protein were upregulated ( P < 0.05) in HP compared with NP. Low-fertility pregnant cows had intermediate endometrial levels of ISG15 mRNA between HP and NP, and ISG15 protein tended ( P < 0.10) to be greater in LP compared with NP cows. This tendency was also observed for increased ISG15 in HP compared with NP in peripheral blood mononuclear cells. Results supported the hypothesis and provided evidence to suggest that Holstein cows with greater DNA-based DGV-DPR values and fewer services per conception have increased conceptus length and enhanced signaling for maternal recognition of pregnancy.

Antioxidant properties and transcriptome of cauda epididymis with different levels of fertility in Hu lambs

Oxidative stress damages the structure of sperm and affects its fertility. Further maturation and storage of spermatozoa occur in the epididymis. Thus, a balanced environment between free-radical production and the antioxidant–enzyme system is important. This study aimed to investigate differences in antioxidative activity in epididymis between high and low fertility levels of Hu sheep. A total of 179 Hu ram lambs aged 56 days were fed up to 6 months in the same environment. After feeding trial, all rams were slaughtered. According to the epididymis weight, cauda sperm concentration, and histo-morphological observation, 18 individuals were selected and divided into low fertility (n = 9) and high fertility (n = 9) groups. The RNA sequencing results showed 1404 differentially expressed genes (DEGs), with 735 genes upregulated and 669 genes downregulated in the high fertility group compared with the low fertility group. Sixteen DEGs related to lipid metabolism and oxidative stress, including GPX3, GPX5, LRP1, PLCB1, CRISP2, BSPH1, and ACSBG1, were screened by qPCR, and results were correlated well with the sequencing data. Antioxidative indices such as total antioxidant capacity (21.29 ± 5.97 vs. 30.23 ± 4.56 μM/gprot) and glutathione peroxidase (39.75 ± 7.61 vs. 68.84 ± 12.51 U/mgprot) increased in the high fertility group. However, the relative expression of mtDNA and malondialdehyde concentration (0.75 ± 0.15 vs. 0.54 ± 0.10 nmol/mgprot) and the reactive oxygen species (ROS) content significantly decreased (P < 0.05). Therefore, epididymis from high-fertility group had strong antioxidative activity and protected spermatozoa from ROS.

First population study of Caribitroides (Caribitroides) tuxtlensis (Amphipoda: Talitridae) from a rainforest in Veracruz state, eastern Mexico

ABSTRACT The present study represents the first effort to analyse the growth, abundance, sex ratio, fecundity and population structure of the terrestrial amphipod Caribitroides (Caribitroides) tuxtlensis from the Santa Martha rainforest, Los Tuxtlas, Veracruz, eastern Mexico. The amphipods were collected using several necro traps during 2001 and were subsequently classified into juveniles, males, females and ovigerous females. The total length of each specimen was measured and each ovigerous female’s eggs were counted. Each individual’s growth and number of modal classes were obtained using Bhattacharya’s method and their growth parameters using the von Bertalanffy equation. A total of 777 specimens were counted, of which 58% were juveniles, 35% were females and 7% were males. A sex ratio of 4.89 females per male was estimated. Four modal classes were estimated based on analysing the length frequencies. The maximum lengths were 13.67 and 13.15 mm and the growth rates were 0.45 and 0.39 for females and males, respectively. The average fecundity was 3.32 ± 1.89 eggs, ranging from one egg in 6-mm females to nine eggs in 12-mm females. The survival rate was 53.69% for females and 38.33% for males. The high percentage of juveniles, the four age classes identified and the sizes obtained are consistent with other terrestrial amphipod species. Based on the number of eggs of reproductive females, this species can be classified within the low-fertility group of amphipods.

An ex-vivo assessment of differential sperm transport in the female reproductive tract between high and low fertility bulls

Despite passing all quality control checks at animal breeding centres, bulls with apparently normal semen quality can yield unacceptably low field fertility rates. This study took an ex-vivo approach to assess if bulls of divergent field fertility differ in the ability of their spermatozoa to interact with the female reproductive tract and its secretions. Six high and six low fertility Holstein Friesian bulls (+4.0 ± 0.2 and −15.7 ± 3.13, respectively; adjusted mean fertility ± s.e.m. mean of the bull population was 0) were selected from a population of 840 bulls with >500 field inseminations per bull. Thawed spermatozoa from each bull were analysed across a range of in vitro assays to assess their ability to transverse the female reproductive tract including; motility and kinematic parameters using computer-assisted sperm analysis, viability, membrane fluidity and acrosomal integrity using flow cytometry as well as mucus penetration tests, rheotactic behaviour and sperm binding ability to the oviductal epithelium. While there was no significant difference between high and low fertility bulls in most of the sperm motility, kinematic and sperm functional parameters (namely, motility, average path velocity, linearity, straightness, amplitude of lateral head movement), viability, membrane fluidity or acrosome intactness, high fertility bulls had higher curvilinear velocity compared to the low fertility group (P < 0.05) and a higher straight-line velocity was observed although it did not reach statistical significance (P = 0.08). There was no difference between treatment groups in the ability of spermatozoa to penetrate periovulatory cervical mucus or in their rheotactic response (P > 0.05). Interestingly, there was a positive correlation between the straight-line velocity of spermatozoa and their rheotactic response (r = 0.45, P < 0.001) and further linear regression analysis indicated 18.9% of the variance in sperm rheotaxis was accounted for by straight line velocity. A higher number of spermatozoa from the high fertility group compared to the low fertility group bound to oviductal explants (15.1 ± 0.98 and 12.5 ± 0.76, respectively; mean ± s.e.m; P < 0.05). In conclusion, the differences in the kinematics of sperm motility and ability to bind to oviductal explants between high and low fertility bulls were modest and are unlikely to explain the inherent differences in fertility between these cohorts of bulls.