Boar Fertility Research Articles

Temporal effect of flaxseed oil in boar’s diet on semen quality, antioxidant status and in-vivo fertility under hot humid sub-tropical condition

The present study investigates the temporal effects of flaxseed supplementation on boar semen quality, antioxidant status, and in-vivo fertility under high-temperature humidity index (THI) conditions in a sub-tropical climate. Twelve Hampshire crossbreed boars were randomly assigned to control and treatment groups, with the treatment group receiving flaxseed oil supplementation. Semen samples were collected and analyzed for semen quality parameters, sperm kinematics, and antioxidant status. Fertility outcomes were assessed through in-vivo mating trials. Flaxseed supplementation resulted in time dependent significant improvements in semen volume, sperm concentration, total and progressive sperm motility, sperm quality parameters, and antioxidant status. Fertility outcomes, including farrowing rates and litter sizes, were also enhanced in the flaxseed-supplemented group. These findings highlight the potential of flaxseed supplementation to improve boar fertility under high ambient stress conditions, with implications for optimizing reproductive performance in swine production systems.

80. Characterization of an in vitro model of sperm storage in pigs: Towards a better prediction of boar fertility?

80. Characterization of an in vitro model of sperm storage in pigs: Towards a better prediction of boar fertility?

Characterization of sexual maturity-associated N6-methyladenosine in boar testes

BackgroundThe health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated.ResultsUsing the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes.ConclusionThe results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Effects of different ages on frozen semen quality and in vitro fertilization efficiency in Wannan black pigs.

According to previous studies, the quality and fertilization rate of fresh sperm from boars of different ages were significantly different. However, the difference of freeze-thaw sperm quality and fertility in boars of different ages is unclear. In this study, boars of a Chinese native breed were assigned into two groups. Each group consisted of five boars aged aged either 2-3 years (young boars = YB) or 5-6 years (aging boars = AB) A total of 60 ejaculates for each group were collected and cryopreserved. Semen quality and in vitro fertility of post-thaw sperm was evaluated. The results showed that the concentration and motility of fresh sperm collected from AB were similar to YB, but their semen volume was higher than that in YB (p < 0.05). Frozen-thawed sperm of AB had lower viability than YB, and higher abnormal rate and reactive oxygen species (ROS) levels of YB (p < 0.05). There was no effect of the age on post-thaw sperm motility and time survival. Functional assessments indicated that increasing age markedly compromises the integrity of the sperm plasma membrane and acrosome, as well as mitochondrial functionality post-thaw, albeit without affecting DNA integrity. Furthermore, increasing age of boars reduces the ability of sperm to bind to the oocyte zona pellucida after thawing, delaying the time of the first embryo cleavage after fertilization. Finally, the early developmental efficiency of in vitro fertilized embryos progressing from 4-cell to blastocyst derived from post-thaw sperm in AB significantly decreased compared to those from YB (p < 0.05). Taken together, these results suggest that increasing age in boars impairs the quality and in vitro fertility of frozen thawed sperm.

Motility-related microRNAs identified in pig seminal plasma exosomes by high-throughput small RNA sequencing

Boar fertility is a key determinant of the production efficiency of the whole pig breeding industry and boar sperm motility is the seminal parameter with the greatest impact on the fecundity of a sow. Exosomes are small, extracellular vesicles found in many body fluids. Seminal plasma exosomes, which are secreted by the epididymis, prostate, seminal vesicles, and testes, contain a large number of miRNAs, the types and levels of which can reflect the physiological state of source cells. It has been shown that the expression profile of seminal plasma exosomal miRNA differs between low-motility semen and normal semen. The aim of this study was to investigate the relationship between semen motility and exosomal miRNA profiles to obtain information that would allow to predict boar fertility, as well as contribute to the understanding of the mechanisms by which exosomal miRNAs regulate semen motility. Three high-motility (semen motility >90 %) and three low-motility (semen motility <80 %) semen samples were collected from Landrace and Yorkshire boars, respectively, and seminal plasma exosomes were extracted by ultracentrifugation. Exosome characterization was performed using transmission electron microscopy, NTA, and Western blot. The expression profiles of exosomal miRNAs associated with semen motility in the two boar breeds were subsequently determined by small RNA sequencing. The results showed that 297 known miRNAs and 295 novel RNAs were co-expressed in the four groups. Notably, six miRNAs (ssc-miR-122–5p, ssc-miR-486, ssc-miR-451, ssc-miR-345–3p, ssc-miR-362, and ssc-miR-500–5p) were found to be differentially expressed in both boar breeds. Enrichment analysis of the target genes of the differentially expressed miRNAs showed that they were mainly involved in biological processes such as regulation of transcription from RNA polymerase II promoter, regulation of gene expression, and intracellular signal transduction and signaling pathways such as the PI3K-Akt, MAPK, and Ras signaling pathways. The six differentially expressed miRNAs identified in this study have significant potential as noninvasive markers of boar semen motility. Meanwhile, the results of the enrichment analysis provide novel insights into the mechanisms underlying the regulation of semen motility.

103 In Utero Exposure to Maternal Heat Stress Impairs Leydig Cell Development and Function in the Boar

Abstract Due to the nearly exclusive use of artificial insemination, a typical U.S. boar sires ~8,398 progeny per year which is a 99.7% greater gene transmission rate compared with a sow (~23.1 progeny/year). Therefore, the boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, boar fertility is threatened by climate change. While boar studs are cooled to prevent heat stress, it is cost prohibitive to efficiently cool the six million pregnant sows that are exposed annually, leading to in utero heat stress (IUHS) of developing progeny. The IUHS of the boar reduces the number of pre-spermatogonia in neonatal testes and decimates subsequent semen quality by reducing both total sperm count (24% decrease) and increasing the proportion of abnormal sperm (42% increase). These data suggest that IUHS programs a lifetime of testis dysfunction reducing the breeding potential of boars. However, the biological mechanisms mediating this effect are undefined. The objective of this study was to determine how IUHS affects Leydig cell development and the testicular proteome. Pregnant sows were exposed to either heat stress (28 to 38°C; n = 18) or thermal neutral (17 to 22 °C; n = 18) conditions from day 27 to 57 of gestation. Neonatal boars exposed to IUHS (n = 23) or in utero thermal neutral (IUTN; n = 29) conditions were castrated at 3 days of age. Testes were preserved for histological analysis and proteomics. Leydig cells within IUHS testes were hypertrophic compared with IUTN controls (147.3 ± 1.6 µm2 versus 132.8 ± 1.4 µm2; P &amp;lt; 0.0001). Moreover, IUHS decreased the number of Leydig cells (Per microscopy field) compared with IUTN males (72.7 ± 2.0 Leydig cells/field versus 93.5 ± 1.8 Leydig cells/field). The testicular proteome was interrogated in a subset of animals (n = 8/treatment). A total of 519 proteins (32% of those detected) were differentially abundant between IUHS and IUTN testes (278 proteins down-regulated, 241 proteins up-regulated) including proteins important for Sertoli (e.g., insulin-like growth factor binding protein), Leydig (e.g., CYP19), and germ (e.g., glyceraldehyde 3-phosphate dehydrogenase) cell function. Notably, gene ontology analysis revealed that both steroid &amp; cholesterol biosynthesis pathways were enriched (P &amp;lt; 0.0001) indicating differential expression of proteins needed for testicular steroidogenesis. Notably, CYP19 (enzyme expressed in porcine Leydig cells that converts testosterone to 17β-estradiol) was down-regulated in IUHS testes. Therefore, these data suggest that IUHS leads to Leydig cell dysfunction in boars. The importance of Leydig cells to male reproduction is unequivocal; thus, IUHS represents a mounting threat to male fertility, especially in the face of climate change.

Selenium Nanoparticles as Potential Antioxidants to Improve Semen Quality in Boars.

Selenium is an essential compound which can influence the fertility of boars by a greater margin. In past decades, research was mainly focused on a bioavailability of various selenium forms and the effect on semen quality. Recently, nanotechnology has expanded the possibilities of selenium supplementation research. Twenty-one Duroc boars (three groups with seven boars each) were included in this experiment with the first group being a control group with no selenium supplementation, and the second group being supplemented with 0.3 mg Se/kg of selenium in inorganic form of Na2SeO3. The third group was supplemented with selenium nanoparticles (100 nm) at the same dose as that of the second group. The experiment lasted for 126 days (three spermatogenesis cycles of boars) and the antioxidant parameters of boar semen were analysed at 42, 84 and 126 days, respectively. The antioxidant parameters (DPPH, FRAP, DMPD, GSH, GSSG) were not influenced by both Se2NO3 and selenium nanoparticle supplementation during this experiment. At the end of the monitored period, significantly higher (p < 0.004) antioxidant readings were observed by using the ABTS method but not the DPPH, DMPD and FRAP methods on the supplemented groups compared to the control. Moreover, selenium-nanoparticle-supplemented groups showed elevated glutathione peroxidase activity in the seminal fluid (p < 0.008). However, the selenium nanoparticle supplementation has not shown an improving effect on sperm quality. This could be considered as a safe alternative to inorganic selenium as well as having a potential to enhance the antioxidant properties of the semen of boars.

Boar fertility is controlled through systematic changes of mitochondrial protein expression during sperm capacitation

Vigorous activation of mitochondria in spermatozoa during capacitation induces the biological and morphological changes of spermatozoa to acquire fertilizing ability. To in-depth understand the dynamic roles of mitochondrial and male fertility, this study was to identify how the mitochondrial proteins are changed during sperm capacitation and regulate male fertility using boar spermatozoa. The mitochondrial proteins were differentially changed during sperm capacitation according to fertility status, i.e., superior litter size (SL) and normal litter size (NL). Following sperm capacitation, ubiquitin-cytochrome c reductase core protein (UQCRC1) and ATP synthase F1 (ATP5F1) increased in NL, while cytochrome c oxidase subunit 5B (COX5B), and cytochrome c1 (CYC1) proteins decreased. In contrast, only and ubiquinone oxidoreductase core subunit 8 (NDUFS8) protein was increased in SL following capacitation. The protein expression difference value of CYC1, COX5B, and NDUFS8 following sperm capacitation was lower in NL than SL boars. Based on these complicated changes during sperm capacitation, the accuracy for predicting male fertility of NDUFS8 was increased to 87 %. Overall, considering the systematic orchestration of mitochondrial protein expression according to sperm capacitation status, it will be possible to better understand male fertility.

Sperm capacitation as a predictor of boar fertility.

Prediction of a boar's fertility level has great economic importance for sow herds. After standard sperm morphology and motility metrics are met, approximately 25% of boars have less than 80% conception rates. Due in part to the many factors involved in the fertilization process, a multifactorial model incorporating multiple relevant sperm physiology factors will likely lead to increased understanding of boar fertility. Here we review the current literature on boar sperm capacitation as a predictor of boar fertility. While limited, several studies have provided correlations between the percentage of sperm in an ejaculate that are capable of undergoing sperm capacitation in a chemically defined media and artificial insemination field fertility as well as proteome and other methods. Work summarized here underscores the need for further understanding of boar fertility.

Cluster analysis and potential influencing factors of boars with different fertility

The fertility of boars is intimately tied to the pig farm's economic benefits. This study aimed to rapidly categorize boars of different fertility and investigate the factors influencing the categorization using the production data in a large pig farm in northern China, including 11,163 semen collection records of Yorkshire boars (215), 11,163 breeding records and 8770 records of farrowing performance of Yorkshire sows (4505), as well as 4720 records of selection indices (sire line index and dam line index) for boars and sows (215 and 4505) between 2017 and 2020. The boar population was classified by two-step cluster analysis, followed by factor analysis to minimize the dimensionality of data variables and eliminate multicollinearity, and then using ordinal logistic regression model to investigate the risk variables impacting boar fertility categorization. Results showed that the two-step clustering divided the 215 boars into three subgroups: high-fertility (n = 61, 28.4%), medium-fertility (n = 127, 59.1%) and low-fertility (n = 27, 12.6%). The high-fertility boars were shown to be substantially greater than the medium-fertility or low-fertility boars (p < 0.05) in average total litter size, number of born alive, and number of healthy piglets of mated sows. Compared with low-fertility boars, the high-fertility boars were also significantly higher (p < 0.05) in the pregnancy rate and farrowing rate of mated sows. However, the three boar subgroups showed no difference (p > 0.05) in semen quality information (average sperm motility, average sperm density, and average sperm volume). Collinearity diagnosis indicated severe multicollinearity among the 20 data variables, which were reduced to 8 factor variables (factors 1–8) by factor analysis, and further collinearity diagnosis exhibited no multicollinearity among the 8 factor variables. Ordered logistic regression analysis revealed a significant and positive correlation (p < 0.05) of boar fertility with factor 2 (average total litter size, number of born alive, number of healthy piglets), factor 4 (average number of weak piglets and average weak piglet rate), factor 6 (sire line index of boars and dam line index of boars), factor 8 (pregnancy rate and farrowing rate), highlighting factor 2 as the most important factor influencing the classification of boar fertility. Our results indicate that the two-step cluster analysis can be used as a simple and effective method to screen boars with different fertility and that farm producers should pay attention to the recording of the reproductive performance of the mated sows due to its role as the risk factor for classification of boar fertility.

Analysis of artificial insemination center management factors that contribute to sperm parameters and boar longevity with a major focus on PGF2α treatment.

Artificial insemination (AI) centers' economic profitability is directly impacted by the amount of high-quality sperm doses. Many internal and external factors contribute to the quality of ejaculates. To enhance the libido and to reduce the time that is required to train boars for semen collection, prostaglandin products are used routinely at AI centers. The objectives of this study were to analyze sperm parameters of 40,765 ejaculates and the survival of 406 PGF2α-treated and 417 untreated AI boars of five breeds (Duroc [DU], Large White [LW], Landrace [LR], Pietrain [PI], and Large White Sire Line [LS]). Data were assessed in one AI center in Switzerland from 2018 to 2022 and sperm parameters were determined by computer-assisted sperm analysis immediately after semen collection. For further calculations, the ejaculates were divided into four groups depending on the boar's age at semen collection day: 7 to 11, 12 to 18, 19 to 24, and 25 to 87 mo. Along with the boar age, breed, season, and semen collection frequency even the treatment with PGF2α had significant influence on sperm parameters. Overall, 19.5% ejaculates were collected after PGF2α treatment. For every age and breed group, higher ejaculate volume was found for untreated boars than for PGF2α-treated boars (P < 0.001). Higher values for total and progressive sperm motility, total sperm number, and the proportion of morphologically normal sperm were observed in untreated boars across several age and breed groups. Only for sperm concentration, higher values were found for treated boars older than 11 mo (P < 0.001) and the breeds DU, LW, and LR (P < 0.001). The chance of ejaculates being rejected for AI purposes because of low sperm quality was 21% greater for treated boars. There was no difference in survival between treated and untreated boars. The hazard for boar removal increased with decreasing age at first semen collection and decreasing semen collection frequency. The results reveal better sperm quality for untreated boars than for PGF2α-treated boars, which might indicate that the underlying fertility of boars necessitating treatment is inferior compared to boars that can be collected without further treatment. Therefore, AI boar stations that are aiming to reduce PGF2α treatment should pay special attention to high libido and fertility of young prospective AI boars.

The effect of gossypol acetate on the fertility of boars

&#x0D; This study evaluated the effect of gossypol acetate, a potential antifertility compound, on semen quality and testicular histopathology of boars. Six boars were allotted into two experimental groups. Half of the animals were fed a diet that was supplemented with 2 mg of gossypol acetate per kg body weight per day for 9 weeks, control animals received a gossypol acetate-free feed. During the experimental period, semen was collected weekly from each boar and semen parameters were recorded. The animals were then euthanized, testicular samples were collected and histopathological examination of the testicular cells, as well as morphometrical analysis of the seminiferous tubules, were performed. The percentage of spermatozoa showing tail abnormalities increased significantly (P=0.017) in the semen of boars fed gossypol acetate-supplemented feed, while several other semen parameters deteriorated without showing statistical significance. Gossypol acetate supplementation also led to a decrease (P=0.042) in the number of spermatogonia in the seminiferous tubules and an increase (P=0.020) in the number of vacuoles in the seminiferous epithelium, consistent with changes seen in cases of male reproductive toxicity. In conclusion, gossypol acetate negatively affected a number of semen characteristics and also had detrimental effects on the histopathology of the testes.&#x0D;

Incorporating A.I. [Artificial Intelligence] into Boar Semen A.I. [Artificial Insemination

Analysis of the U.S swine herd shows variation in pregnancy rate is more attributable to male- factor subfertility than the dam. To date, a limited degree of correlations has been observed between conventional semen analysis parameters and actual fertility after standard quality cutoffs are met. Thus, a clear ability to predict male-factor fertility is lacking. Recent technological advances in flow cytometry, namely image-based flow cytometry, allows for high-throughput, single-cell phenotyping. This allows for multi-million sperm bioimage data sets to be easily attained. These datasets can then be analyzed utilizing machine & deep learning analytic methods and correlated with single sire field fertility data to open the black box of boar fertility prediction. In this presentation, we will discuss recent advancements our lab has made utilizing machine & deep learning, artificial intelligence into boar sperm quality analysis. This includes bioimage, deep learning analysis of boar sperm morphology as well as our latest results related to boar semen fertility prediction. Our findings establish a new era for boar sperm quality analysis and pave the way for accurate fertility prediction in future precision agriculture applications. Work performed here was supported by USDA’s National Institute of Food and Agriculture grant number 2019- 67012-29714 (KK) and 2022-67015-36298 (KK).

PSV-A-7 Quality Assessments of Sperm from Gnrh-II Receptor Knockdown (KD) Boars

Abstract Pigs are the only livestock species encoding functional proteins for both the second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). Previously, we determined that GnRHR-II localizes to the connecting piece of ejaculated sperm, whereas GnRH-II is present within seminal plasma, suggesting an autocrine/paracrine interaction influencing porcine sperm function. To examine the GnRH-II/GnRHR-II system further, we produced a swine line with reduced endogenous expression of GnRHR-II (GnRHR-II KD). Therefore, our objective was to assess sperm quality of ejaculates collected from GnRHR-II KD (n = 3) and littermate control (n = 3) boars. Semen was extended (37.5 x 106 sperm/mL) in Androstar Plus and subjected to flow cytometry (Kuster Research and Consulting, Inc.; Atkinson, IL). Assays measured DNA compaction, membrane fluidity and viability, acrosome integrity, mitochondrial depolarization, and morphology. Data were analyzed using the MIXED procedure of SAS; the model included ejaculate as the experimental unit and line as a fixed effect. DNA compaction, membrane fluidity, acrosome integrity, mitochondrial depolarization, and morphology were not statistically different between lines (P &amp;gt; 0.05). However, the percentage of sperm with a viable (intact) plasma membrane was reduced in GnRHR-II KD boars (87.4 ± 0.8%) compared with controls (89.6 ± 0.8%; P &amp;lt; 0.05). Although differences did not reach significance, DNA compaction (a measure of sperm chromatin integrity when undergoing acid stress) was doubled and mitochondrial depolarization (reflective of mitochondrial function) was 11% greater in sperm from transgenic males relative to littermate controls, indicating that GnRHR-II KD sperm are more susceptible to environmental stressors and have altered energy production. Thus, these initial data suggest disruptions in the function and fertilizing capacity of GnRHR-II KD sperm, implying that GnRHR-II may represent a potential biomarker for boar fertility. Supported by USDA/NIFA AFRI (2017-67015-26508) and Hatch Multistate (NEB-26-244) funds. USDA is an equal opportunity provider and employer.

Integrity Analysis and Functional Sperm Assessments to Predict Boar Fertility Performance

Integrity Analysis and Functional Sperm Assessments to Predict Boar Fertility Performance

Birthweight leads to seminal and testicular morphofunctional commitment in sexually mature boars

Given the importance of males as semen donors for artificial insemination (AI) and the high incidence of low birthweight piglets at commercial farms, the impact of birthweight on fertility in boars deserves special attention. The aim of this study was to evaluate testicular morphofunctional parameters and semen characteristics in different birthweight boars. Forty littermate males were selected at birth and divided into two experimental groups, according to birthweight: high (HW, birthweight ranging from 1.80 to 2.15 kg, n = 20) and low (LW, birthweight ranging from 0.75 to 1.10 kg, n = 20). At 170 days of age, a sub-set of 24 littermate boars (n = 12 HW and n = 12 LW) were randomly selected for semen collection, which was performed once a week, at a 15-day interval, during five weeks. At 300 days of age, boars were orchiectomized, and the testis processed for histological and molecular analyses. The HW group was heavier and presented larger testes compared to LW animals (P < 0.05). Despite that, birthweight did not significantly affect semen volume or sperm quality parameters (concentration, motility, vigor or morphology), although LW boars produced 38.2% fewer total sperm and 24% lower semen concentration, leading to 36.8% less inseminating doses. The histomorphometrical evaluation showed that seminiferous tubules diameter and germinal epithelium height were similar between experimental groups. However, LW boars presented shorter seminiferous tubules and, consequently, fewer Sertoli cells per testis (P 0.05). Even though plasma testosterone levels were equivalent in both birthweight groups, LW testis presented less androgen receptors (P < 0.05). Additionally, birthweight was positively correlated with total seminiferous tubule length and number of Sertoli cells (P < 0.01), and with body and testis weights (P < 0.01). Taken together, even though adult LW boars showed no evident semen pathologies or spermatogenesis commitment, mature HW males have the potential to produce more spermatozoa, consequently more semen doses per ejaculate, being more valuable to an industry that relies on AI.

20 Trials and Tribulations of Countering Insufficient Sperm Quantity with Sperm Quality, Fertility, Synchrony and Deeper Site of Insemination

Abstract Superior tools and techniques for boar semen evaluation, semen preservation and artificial insemination (AI) have helped build industry confidence and competence to reduce sperm per female inseminated without compromising reproductive results. Over the last 30 years, the average number of sperm per AI dose has decreased from about 5 to 2 billion in the US and doses containing 1.5 billion sperm have become common in some other countries. While even more genetic lag can be eliminated by further reduction of sperm dose, the level at which fecundity and fertility loss occurs is not a constant and likely varies between different farms and production systems. In general, uterine delivered sperm doses near or lower than 1 billion tend to reduce litter size and further sperm dose reduction eventually reduces pregnancy rate. Deeper catheter deposition of semen, beyond the uterine body, does not seem to effectively compensate for a limiting quantity of sperm. The exception is that surgical injection of sperm into the oviducts requires much less sperm than intrauterine insemination. Reduction of the AI-to-ovulation interval, from 24 to 8 h for example, does not seem to ameliorate the negative effects of a limiting number of sperm when sperm quality is high. The exception may be that a shorter AI-to-ovulation interval can increase reproductive results when both sperm quantity and quality are limiting. Examples include low boar fertility, advanced semen age, or sperm quality compromised by cryopreservation or sex-sorting. A better understating of boar fertility, the interaction of sub-populations of sperm with the oviductal reservoir and their participation in fertilization is likely requisite to continued reduction of the number of sperm required per female. Technologies that predict or regulate sperm quality, fertility and ovulation could prove useful in the quest to use sperm more efficiently and add value to swine AI.

MiRNA-Profiling in Ejaculated and Epididymal Pig Spermatozoa and Their Relation to Fertility after Artificial Insemination.

Simple SummaryThe present study searched for the presence and abundance of porcine spermatozoa small RNA sequences (microRNAs) that have the potential to alter gene expression patterns. Four different sperm sources were compared: spermatozoa from three different sections of the ejaculate and from the caudal epididymis, also classed as spermatozoa from higher (HF) or lower (LF) fertility boars. Sperm miRNAs were compared using high-output small RNA sequencing. We identified five sperm miRNAs not previously reported in pigs. Differences in abundance of four miRNAs known to affect the expression of genes with key roles in fertility were related to boar fertility. These miRNAs could be used as fertility markers in artificial insemination programs.MicroRNAs (miRNAs) are short non-coding RNAs (20–25 nucleotides in length) capable of regulating gene expression by binding -fully or partially- to the 3’-UTR of target messenger RNA (mRNA). To date, several studies have investigated the role of sperm miRNAs in spermatogenesis and their remaining presence toward fertilization and early embryo development. However, little is known about the miRNA cargo in the different sperm sources and their possible implications in boar fertility. Here, we characterized the differential abundance of miRNAs in spermatozoa from the terminal segment of the epididymis and three different fractions of the pig ejaculate (sperm-peak, sperm-rich, and post-sperm rich) comparing breeding boars with higher (HF) and lower (LF) fertility after artificial insemination (AI) using high-output small RNA sequencing. We identified five sperm miRNAs that, to our knowledge, have not been previously reported in pigs (mir-10386, mir-10390, mir-6516, mir-9788-1, and mir-9788-2). Additionally, four miRNAs (mir-1285, mir-92a, mir-34c, mir-30), were differentially expressed among spermatozoa sourced from ejaculate fractions and the cauda epididymis, and also different abundance was found between HF and LF groups in mir-182, mir-1285, mir-191, and mir-96. These miRNAs target genes with key roles in fertility, sperm survival, immune tolerance, or cell cycle regulation, among others. Linking the current findings with the expression of specific sperm proteins would help predict fertility in future AI-sires.

483 Current Knowledge Gaps in Swine Reproduction

Abstract Identifying swine reproduction research opportunities, especially those that add value by addressing industry challenges and leveraging assisted reproductive technologies (ART) will be the focus of this talk. ART and genetic selection to increase pork production efficiency have been intertwined since the use of artificial insemination (AI) became widely adopted 30 years ago. Tremendous efficiency gains have been made in part by increasing litter size which is nearly 6 to 7 pigs larger today than it was in 1990. The increased farrowing duration and stillbirths, and decreased piglet birth weight, colostrum intake, and survival to weaning that has been associated with these larger litters are prime targets to be ameliorated with sow and pig management solutions. There is also clear evidence that pigs from smaller litters have the advantage of superior reproductive performance compared to those from larger litters. Thus, litter size and pre-weaning growth may need to be managed, or at least taken into account, to select the best boars and gilts for breeding stock and replacements. Any other accurate, early indicators of boar and gilt puberty, fertility, and lifetime productivity could add significant value. The seasonal fluctuation in fertility that has plagued pig production is costly and may be smoothed out by strategies that mitigate heat stress and improve health. Another opportunity to improve both pig and sow welfare may be intermittent suckling and induction of a fertile estrus and ovulation for breeding during lactation. Even more genetic and pig growth value could be added via AI with sex-sorted semen, if methods to better estimate boar fertility, deliver increased sperm quality, and more precisely synchronize or predict ovulation can be found to reduce the number of sperm required per female. Existing and evolving ART will synergize with genomics and gene editing technologies to unlock new levels of production efficiency.

14 Opening the Black Box of Fertility Prediction

Abstract Analysis of the U.S. swine herd shows variation in pregnancy rate is more attributable to male-factor subfertility than the dam. To date, a limited degree of correlations has been observed between conventional semen analysis parameters and actual fertility after standard quality cutoffs are met. Thus, a clear ability to predict male-factor fertility is lacking. Knowledge of what makes fertilization competent spermatozoa has been long sought after for centuries. It was only in the last half-century that we understood spermatozoa undergo a biological process after ejaculation to acquire the capacity to fertilize. Since then, work has been done to elucidate the molecular pathways involved in sperm capacitation. Recent technological advances in flow cytometry, namely image-based flow cytometry, allows for high-throughput, single-cell phenotyping. Single-cell phenotyping with biomarkers reflecting significant sperm capacitation events, mitochondrial status, cell health, and more, allows multi-million bioimage data sets to be easily attained. These datasets can then be analyzed utilizing machine and deep learning analytic methods and correlated with single sire field fertility data to open the black box of boar fertility prediction. Our findings establish a new paradigm in sperm function and pave the way for accurate fertility prediction in future precision agriculture applications. This work was supported by the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture (USDA) award number 2019-67012-29714.