Ejaculate Collection Research Articles

240 Impact of structural traits on semen collection training and lesion characteristics in Duroc boars

Abstract Rising incidence of lameness in boar stud contributes to increased morbidity and mortality, affecting longevity and likely semen output. Lameness is not unique to the boar and affects the swine industry as a whole, contributing to millions of dollars in losses every year. Hypothesized contributors to lameness incidence are body, feet, and leg structure. In the boar stud lameness affects the ability of the boar to mount the dummy and can cause pain-related stress which can impact reproductive function. Lameness is also often accompanied by skin lesions or wounds which can lead to infection and further semen production. Our objectives were to evaluate the relationship between boar structural traits and semen collection training traits and determine if structural traits influence presence and severity of skin lesions. Structural traits of developing Duroc boars (n = 82; 190 ± 10.3 d of age) were assessed at arrival to the stud and 6 wk later. Structural traits assessed included front and rear leg set, foot shape, chest width, and hip width (1 = poor; 5 = ideal). An overall structure score for each boar was determined by adding all structural trait scores. Lesions were assessed on a zero to three scale (0 = no lesions; 3 = severe lesions). Based on overall structure score, boars were categorized into one of three groups (poor, intermediate, or ideal) to determine if structural traits affected the presence and severity of lesions. All boars were subjected to semen collection training 1 wk after arriving at the stud and considered trained when successful mounting and ejaculation occurred. Pearson’s correlation coefficients were determined between total structure score, lesion severity, and semen collection training characteristics. The GLIMMIX procedure of SAS was used to assess lesion presence differences between structure groups. Structure score and lesion severity were not significantly correlated (-0.017; P = 0.91) with number of attempts to train, time to mount, or time to collection of ejaculates. Upon arrival at the stud, boars in the poor structure group displayed a greater (P < 0.01) incidence of lesions than intermediate and Ideal structure groups but did not have greater lesion severity (P = 0.74). After 6 wk in the boar stud, there were no differences (P = 0.83) in lesion presence or severity between structure groups, suggesting the lameness issues are more likely caused by housing environment or other factors than structural traits.

#110 : Diagnostic Approach and Clinical Recommendations for Non-obstructive Azoospermia: Global Andrology Forum Survey Results

Background and Aims: Non-obstructive azoospermia (NOA) is the most common form of male infertility, characterized by an absence of sperm in ejaculate and is caused by testicular failure. While NOA afflicts 10-38.3% of infertile males, there is no specific recommendations in EAA/EAU guidelines on its clinical diagnosis or WHO guidelines on semen analysis. Therefore, we conducted a global survey to assess the standard approach of practitioners in the diagnosis and management of NOA. Method: The Global Andrology Forum (GAF) conducted a survey on 436 independent practicing entities from 55 countries. We compiled data from 336 respondents (andrologists, urologists, fertility specialists). Information collected included frequency of NOA, varicocele, frequency of obtaining semen, interval between obtaining semen samples, hormone evaluation (FSH, LH, prolactin, total/free testosterone, SHBG, estradiol, inhibin), genetic testing (karyotype, Y-chromosome deletion, CFTR, Kal1) and referral to genetic counsellors. Results: The majority of participants (80%) either reported <10% or 10-25% NOA frequency in their population (Fig. 1A). 40.6% of participants stated that 10-25% of men with NOA cases have a clinically significant varicocele (Fig. 1B). For NOA diagnosis, majority of participants cited the requirement of at least two semen samples, with a period of 14-29 days as the interval between two semen analyses. To achieve high-quality semen, consideration must be given to an abstinence period (2-7days) and the complete collection of ejaculates. The survey reported FSH (97%), LH (86.9%) and total testosterone (92.9%) tests as most frequently used for NOA patients (Fig. 2A). The initial hormonal evaluation of a patient with suspected NOA should include total testosterone levels and LH, as these hormones are the primary regulators of spermatogenesis. Karyotype analysis (86.2%), Y-microdeletion (88.3%), CFTR (21.9%) and Kal1 (2.7%) were most routinely performed genetic tests (Fig. 2B). Referrals to genetic counsellors is recommended as these defects have the potential to be passed to future generations. Conclusion: This global study evaluates NOA frequency, clinical diagnosis and management by specialists, in an effort to fill the gap in NOA research. Concomitantly, we provide GAF expert recommendations to enhance our collective understanding of NOA, optimize patient care and improve the management of NOA.

Reaction time as a libido indicator and its relation to pre-freeze and post-thaw sperm quality in bulls.

Libido and sperm quality output relationship is already not clear in farm animals. The present study compared reaction time (RT) as a libido indicator and the pre-freeze and post-thaw sperm quality of AI bulls. Before the collection of ejaculates (n = 53, from 22 AI bulls [4.2 ± 1 years of age]), RTs were collected using a chronometer as the interval between the bull's arrival at the semen collection area and his first false mount (FM) on another male. The ejaculates were examined for their volume, concentration and motility. Subsequently, all aliquots were diluted with a commercial semen extender and equilibrated for 3 h before freezing. Frozen semen samples were thawed and examined for sperm kinematics using CASA, plasma membrane and acrosome integrity of sperm (PMAI) by flow cytometry. Additionally, the temperature humidity index (THI) values were assessed during the study. Multiple linear regression analysis was used to analyse the data. The results indicated that THI had a significant effect on libido (p < .001). However, libido had no effect on either pre- or post-thaw sperm quality parameters except for the velocity of the average pathway (VAP) (p < .05). Therefore, relying solely on RT -libido- as an indicator of bull sperm quality at AI stations may not be reliable, as it is a complex behavioural assessment.

Noninvasive Ejaculate Collection from the African Turquoise Killifish Nothobranchius furzeri.

Ejaculate collection from live animals is a fundamental technique used to perform repeated measures on the same individual-for instance, to test the effect of different treatments/conditions on the same animal or to conduct longitudinal studies. Moreover, collecting the ejaculate allows one to perform in vitro fertilizations (IVFs) or sperm cryopreservation from valuable individuals (e.g., mutant, transgenic animals) without the need to kill the animals. We developed a noninvasive protocol based on a stripping procedure for collecting the ejaculate from live males of the African turquoise killifish (Nothobranchius furzeri). The proposed procedure allows the retrieval of mature sperm along with the seminal fluid. The ejaculate can be used within 1 h of the collection to perform in vitro fertilizations and/or sperm quality measurements. Contamination by mucus, water, urine, or feces can activate the sperm and/or negatively affect sperm quality, so special care is needed to avoid contamination and thus prolong sperm viability. Since in small fish the amount of ejaculate collected is limited to a few microliters, in the present protocol we also provide recommendations on housing conditions to maximize the probability to obtain ejaculate samples.

Curcumin Attenuates Damage to Rooster Spermatozoa Exposed to Selected Uropathogens.

Artificial insemination, as an essential pillar of the modern poultry industry, primarily depends on the quality of semen collected from stud roosters. Since the collection and storage of ejaculates is not a sterile process, antimicrobial agents have become essential supplements to semen extenders. While the use of traditional antibiotics has been challenged because of rising bacterial resistance, natural biomolecules represent an appealing alternative because of their antibacterial and antioxidant properties. As such, this study strived to compare the effects of 50 μmol/L curcumin (CUR) with 31.2 µg/mL kanamycin (KAN) as a conventional antibiotic on rooster sperm quality in the presence of Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa. Changes in sperm structural integrity and functional activity were monitored at 2 and 24 h of culture. Computer-assisted semen analysis revealed significant sperm motility preservation following treatment with KAN, particularly in the case of Salmonella enterica and Pseudomonas aeruginosa (p &lt; 0.001) after 24 h. On the other hand, CUR was more effective in opposing ROS overproduction by all bacteria (p &lt; 0.05), as determined by luminol-based luminometry, and maintained sperm mitochondrial activity (p &lt; 0.001 in the case of Salmonella enterica; p &lt; 0.05 with respect to Escherichia coli and Pseudomonas aeruginosa), as assessed by the fluorometric JC-1 assay. The TUNEL assay revealed that CUR readily preserved the DNA integrity of rooster sperm exposed to Salmonella enterica (p &lt; 0.01) and Escherichia coli (p &lt; 0.001). The bacteriological analysis showed higher efficiency of KAN in preventing the growth of all selected bacterial species (p &lt; 0.0001) as opposed to CUR. In conclusion, CUR provided protection to rooster spermatozoa against alterations caused by uropathogens, most likely through its antioxidant activity. Hence, CUR supplementation to poultry semen extenders in combination with properly selected antibacterial substances may become an interesting strategy in the management of bacterial contamination during semen storage.

Candida Genus Maximum Incidence in Boar Semen Even after Preservation, Is It Not a Risk for AI though?

There is little information in the literature about the fungal contamination of boar semen and its persistence during storage. The challenge of this study was to perform a mycological screening to identify the yeast in the raw semen at 12/24 h after dilution. The research was done in pig farms in the N-E area of Romania, with maximum biosecurity and state-of-the-art technology. All the examined ejaculates (101) were considered to be normal for each spermogram parameter, with microbiological determinations in T0 at the time of ejaculate collection, T1 at the time of dilution, and T2 at 24 h of storage. Microbiological determinations (mycological spermogram) were performed for quantitative (LogCFU/mL) and qualitative (typification of fungal genera) identification. Bacterial burden (×103 LogCFU/mL) after dilution (T1) decreased drastically (p &lt; 0.0001) compared to the one in the raw semen (T0). After 24 h of storage at 17 °C, the mean value of the bacteriospermia remained constant at an average value of 0.44. Mycospermia had a constant trend at T0 (raw) and T1 (0.149 vs. 0.140) and was slightly higher at T2 (0.236). The difference between T1 vs. T2 (p = 0.0419) was close to the statistical reference value (p = 0.05). Of the total genera identified (24), the fungi had a proportion of 37.4% (9/15) and a ratio of 1:1.6. Regarding the total species (34), the fungi had a frequency of 29.42% (10/24) with a ratio between the fungi and bacteria of 1:2.4. A fertility rate of 86% was observed in the L1 group (50 AI sows with doses and mycospermia from T1), and an 82% rate was observed in the L2 group (50 AI sows with doses and mycospermia from T2). The litter size of L1 was 9.63 piglets and 9.56 for L2. Regarding the total number of piglets obtained between the two groups, there was a slight decrease of 22 piglets in group L2, without statistical differences (p &gt; 0.05). The predominant genera persisted after dilution during a 12 h storage at 17 °C, where yeasts, such as Candida parapsilosis and C. sake were identified in more than 92% of AI doses.

Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility

Younger bulls typically produce lower volumes of semen per ejaculate with a lower sperm concentration than older more mature, bulls and often fail to meet semen demand using standard collection frequency schedules. The objective of this study was to assess the effect of ejaculate collection frequency on semen output, sperm quality and field fertility in young bulls under commercial conditions. Holstein Friesian bulls aged 366 ± 8 days (mean ± SEM) were assigned one of two ejaculate collection frequencies: (i) HF (n = 14 bulls), where ejaculates were collected twice a day, five days in each two-week period or (ii) LF (n = 12 bulls), where ejaculates were collected once a day, two days per week. The trial period continued until each bull reached both 20 ejaculates and 1000 marketable frozen semen straws. Subjective motility was assessed on all ejaculates pre-freeze and post-thaw (at 0 and 2 h). A subset of ejaculates were assessed post-thaw by computer-assisted sperm analysis for motility, kinematics and morphological defects and by flow cytometry for viability, membrane fluidity, acrosome integrity, reactive oxygen species and DNA fragmentation. A total of 13,846 inseminations (9,541 for HF and 4,305 for LF) were carried out on dairy cows and heifers. HF reached the 1000 straw threshold 41 days earlier than LF (P < 0.01) with the same number of ejaculates. Ejaculate volume and sperm concentration were not affected by treatment but the first ejaculate of the day (HF only) had a greater volume (P < 0.001) and sperm concentration (P < 0.05) than the second ejaculate. HF had higher pre-freeze total (P < 0.01) and gross (P < 0.05) motility than LF. HF had higher post-thaw (2 h) total and gross motility than LF (P < 0.05). Ejaculate rejection rates did not differ between treatments. There was no effect of treatment, week or ejaculate number of the day (HF only) on post-thaw motility and kinematic parameters or sperm viability, membrane fluidity, acrosome integrity and DNA fragmentation. However, HF had lower superoxide production than LF (P < 0.05). Pregnancy per artificial insemination was 64.5 ± 1.0% and 59.9 ± 1.1% for the HF and LF bulls, respectively (mean ± SEM; P = 0.05). In conclusion, collecting ejaculates more frequently from young bulls significantly reduced the number of days required to obtain 1000 straws, increased semen quality in terms of lower superoxide production and increased field fertility.

Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects.

The aim of the study was to assess changes in the integrity of sperm cell membranes during the storage of semen collected from Duroc × Pietrain crossbred boars and purebred boars of the component breeds. To compare the cell membrane integrity of sperm heads in crossbred and purebred boars, heterosis effects were estimated. The study was conducted on 48 ejaculates collected from Duroc × Pietrain crossbred boars and from purebred Duroc and Pietrain boars used for artificial insemination. Microscope slides were prepared from each ejaculate for the evaluation of the cell membrane integrity of the sperm, at 1, 24, 48, 72, and 96 h after collection of the ejaculate. Diluted ejaculates were stored at 17 °C. Sperm membrane integrity was analysed by two methods: SYBR-14/PI and eosin-nigrosin. Our results showed that the cell membrane integrity of sperm heads changed with storage time, but the extent of the changes varied depending on the genetic group of boars. The semen of Duroc × Pietrain crossbreds was clearly seen to be less sensitive to storage conditions than that of boars of the parent breeds, which was confirmed by the calculated heterosis effects. The percentage of sperm with an intact cell membrane was higher in crossbred boars than in purebred boars (p ≤ 0.05). In addition, significantly fewer moribund sperm spermatozoa and spermatozoa with a damaged cell membrane were observed in crossbred boars (p ≤ 0.05). In the semen of purebred Duroc and Pietrain boars, the cell membrane integrity of the sperm should be assessed more often during storage than in the semen of Duroc × Pietrain crossbred boars. This study provides valuable information for the development and implementation of semen quality monitoring in crossbred boars and boars of the parent breeds during storage at 17 °C with respect to the cell membrane structure of sperm heads. The evaluation methods used effectively identify damage to the cell membranes of the sperm during semen storage.

The Usefulness of Sexual Behaviour Assessment at the Beginning of Service to Predict the Suitability of Boars for Artificial Insemination.

Simple SummaryBoars producing ejaculates of high quality and shortest duration are particularly valuable; this aspect can be the primary factor in inbreeding development and improvement of swine utilization. It is important to develop objective ways to describe the sexual activity of various breeds of boars and its application and effectiveness in conducting artificial insemination. The results of this study indicate that the time from entering the arena until achieving an erection, which is assessed at the beginning of a boar’s breeding utilization, may be used to predict a boar’s future libido. There was an association between the level of libido and ejaculate characteristics. Boars requiring the most time to begin ejaculation produce ejaculates with a higher sperm concentration and number.Parameters of sexual activity were determined in 49 young boars used for artificial insemination, four times at three-month intervals. The parameters included the time from entering the arena until mounting the phantom; the time from mounting the phantom until achieving erection; the time from achieving full erection until the start of ejaculation; duration of ejaculation; and the number of times the boar mounted the phantom. Characteristics of the ejaculates were also assessed. The libido parameter associated with the greatest efficacy of artificial insemination was the effectiveness of artificial insemination service, the time from entering the arena until the start of ejaculation. The significance of this trait for predicting ejaculation performance was analysed. The libido characteristics were classified into three categories: boars with a short reaction time to the phantom, boars with an intermediate reaction time, and boars with a long reaction time. For these groups, the characteristics of ejaculates collected at the start of the period during which ejaculates were collected and after three, six and nine months were determined. The sexual experience of boars was not associated with the expression of sexual behaviour because young boars during their first three months of ejaculate collections required less time to initiate ejaculation. The ejaculates with the greatest utility were obtained after six months of service. These ejaculates had the largest volume (255.22 mL), and the most insemination doses could be prepared from these ejaculates. On average, more than 23 insemination doses were prepared from ejaculates collected after six months of semen collections, which is about four doses more than from ejaculates collected at the start of artificial insemination service (p < 0.01).The time from entering the arena to beginning ejaculation can be used to predict a boar’s future libido. A relationship was shown between the level of libido and ejaculate characteristics. The ejaculates of the boars which needed the longest time to begin ejaculation at the start of semen collections had the greatest sperm concentration and number. In group 3, the boars’ejaculates contained about 6–9 × 109 more sperm than the ejaculates of boars from group 1. After six months of the experimental period, the difference was nearly 15 × 109 sperm (p < 0.05), and after nine months, it exceeded 22 × 109 sperm (p < 0.01).

Aldose Reductase B1 in Pig Seminal Plasma: Identification, Localization in Reproductive Tissues, and Relationship With Quality and Sperm Preservation.

Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.

115. Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility

115. Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model.

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.

Delays in processing and storage of pig seminal plasma alters levels of contained antioxidants

Seminal plasma (SP) antioxidants are considered biomarkers of sperm function and fertility for AI-boars. The current protocol for their measurement implies the SP was harvested immediately after ejaculation and prompt stored at −80 °C until analysis. Such protocol may be impractical for AI-centers. This study evaluated how SP levels of antioxidants were influenced by delays in (1) SP-harvesting (0 [control], 2 or 24 h at 17 °C after ejaculate collection), in (2) SP-freezing (0 [control] or 24 h at 17 °C after SP-harvesting) or (3) the temperature of storage (−80 °C [control] or − 20 °C). The SP-antioxidants evaluated were: glutathione peroxidase [GPx], superoxide dismutase [SOD], paraoxonase-1 [PON-1], trolox equivalent antioxidant capacity [TEAC] and oxidative stress index [OSI]. A total of 120 aliquots from 10 entire ejaculates were handled in three trials. They were centrifuged (1500 g, 10 min) for harvesting SP and antioxidants were measured with an Automatic Chemistry Analyzer. A 24 h-delay in harvesting the SP led to an increase (p˂0.001) in TEAC and SOD SP-levels, and a decrease (p˂0.05) of OSI and PON-1. Similarly, a 24 h-delay to freeze the SP increased (p˂0.01) TEAC values and decreased (p˂0.01) PON-1 and GPx activity levels. Finally, storing the SP at −20 °C decreased (p˂0.001) SP-levels of TEAC, PON-1 and GPx, and increased (p˂0.01) OSI values. Strong positive relationships (p˂0.001) were found between antioxidant SP-levels in processed samples and their respective controls. In sum, handling and SP storage influence antioxidant measurements in AI-boars. Reliable levels of SP-antioxidants can only be warranted if a strict protocol for harvesting and SP storage is followed.

Ejaculate Collection Influences the Salivary Oxytocin Concentrations in Breeding Male Pigs.

Simple SummaryThis study aimed to evaluate how the process of ejaculate collection affects oxytocin concentrations in saliva of boars used in artificial insemination. Saliva samples of 33 boars were collected the day before ejaculate collection, during the ejaculation time, and two hours after ejaculate collection. Free oxytocin and oxytocin linked to proteins were quantified in these saliva samples. Oxytocin concentrations during the ejaculation time were higher than the day before with oxytocin linked to proteins showing higher differences. In addition, younger boars, boars with higher libido intensity and boars of the Pietrain breed showed higher values of oxytocin in saliva during ejaculation than the day before. This study demonstrated that ejaculation influences the salivary oxytocin concentrations boars.The objective of the present study was to evaluate the possible changes of oxytocin concentrations in saliva during and after ejaculate collection in breeding boars usually used in artificial insemination programs. Saliva samples of 33 boars were collected the day before ejaculate collection (DB), during the ejaculation time (T0) and two hours after ejaculate collection (T2). Free oxytocin and oxytocin linked to proteins concentrations were measured by two methods previously developed and validated for saliva of pigs. Younger boars, boars with higher libido intensity and boars of the Pietrain breed showed higher values of oxytocin in saliva during ejaculation than the day before. In addition, boars with higher libido showed higher concentrations two hours after ejaculate collection than during the day before. These changes were of higher magnitude and significance when oxytocin linked to proteins was measured. In conclusion, this study demonstrated for the first time that ejaculation influences the salivary oxytocin concentrations in breeding boars, although this influence varies according to age, libido and breed.

Influence of Collection Frequency on Boar Ejaculate Quality Parameters

The aim of study was to examine the effect of the boar ejaculate collection frequency on the parameters of its quality. The research covered a three-year period, and four most common boar breeds in our area. A total of 1705 ejaculates were taken from Yorkshire boars, 1693 ejaculates from Landrace boars, 1106 ejaculates from Pietrain boars and 669 ejaculates from Duroc boars. Within each breed, groups were formed according to the frequency of ejaculate taking (once, twice, three, four, five, or six times a month). Evaluation of ejaculate quality was performed in a reprocentre laboratory, according to their quality standards. Quality parameters that have been determined were the volume of ejaculate, sperm concentration, progressive sperm motility, live sperm count, the number of doses per ejaculate and the overall assessment of ejaculate. Results of this study show that the volume and progressive motility increase with the increase of frequency in ejaculate taking, while the sperm concentration drops, except for the Pietrain boars. The best results were observed in ejaculates collected 5-6 times during the month. The highest mean volume of ejaculate was found in the Landrace boars (292.22 ml), in ejaculate taking frequency of five times per month. The highest average progressive sperm motility was 92% in the Yorkshire boars, on collection frequency of six times a month, while the highest concentration of spermatozoa was found in the Duroc boars in collection frequency of one taking per month (447.32 mil/ml). The results of this study indicate that the frequency of ejaculate taking affects the boar semen quality parameters.

Exogenous melatonin advances the ram breeding season and increases testicular function

Governed by melatonin, ovine reproductive seasonality limits production outcomes due to periods of decreased reproductive efficiency. Though it is established that slow-release melatonin implants improve out of season reproductive performance in the ewe, the comprehensive effects of exogenous melatonin in the ram remain inconclusive. This study aimed to ultimately clarify the ability of exogenous melatonin to alter ram reproductive function during the non-breeding season and the subsequent breeding season. Hence, we investigated the effect of exogenous melatonin on reproductive endocrinology, semen quality and production, testicular size and libido in Merino and Poll Dorset rams (n = 31, using a subset of 18 rams for analysis of semen production and quality). Melatonin treatment resulted in elevation of melatonin in seminal plasma from 1–8 weeks post-implantation and in blood plasma at 6 weeks post-implantation. The blood plasma testosterone of implanted rams was greater than controls at both 6 weeks post-implantation and during the following breeding season. Implanted rams exhibited increased testicular size and number of sperm per ejaculate from 3–12 weeks post-implantation but did not demonstrate any change in sperm motility or morphology in response to treatment. Compared to their control counterparts, melatonin-treated Poll Dorset rams exhibited a lower percentage of sperm DNA fragmentation during several weeks of the non-breeding season. Though melatonin increased the likelihood of ejaculate collection in Poll Dorset rams (P < 0.05), libido was otherwise unaffected by treatment. Melatonin did not alter seminal plasma concentrations of inhibin A or Anti-Mullerian hormone, however, for the first time in the ram we have shown Anti-Mullerian hormone to be positively correlated with the number of sperm per ejaculate and sperm motility (r = 0.464 and 0.3242 respectively, P < 0.001), and inhibin A to be correlated to the number of sperm per ejaculate (r = 0.1786, P = 0.0135). These results indicate that melatonin is able to both systemically upregulate reproduction and act directly upon testicular function in the ram.

Transcriptome profiling of porcine testis tissue reveals genes related to sperm hyperactive motility

BackgroundSperm hyperactive motility has previously been shown to influence litter size in pigs, but little is known about the underlying biological mechanisms. The aim of this study was to use RNA sequencing to investigate gene expression differences in testis tissue from Landrace and Duroc boars with high and low levels of sperm hyperactive motility. Boars with divergent phenotypes were selected based on their sperm hyperactivity values at the day of ejaculation (day 0) (contrasts (i) and (ii) for Landrace and Duroc, respectively) and on their change in hyperactivity between day 0 and after 96 h liquid storage at 18 °C (contrast (iii)).ResultsRNA sequencing was used to measure gene expression in testis. In Landrace boars, 3219 genes were differentially expressed for contrast (i), whereas 102 genes were differentially expressed for contrast (iii). Forty-one differentially expressed genes were identified in both contrasts, suggesting a functional role of these genes in hyperactivity regardless of storage. Zinc finger DNLZ was the most up-regulated gene in contrasts (i) and (iii), whereas the most significant differentially expressed gene for the two contrasts were ADP ribosylation factor ARFGAP1 and solute carrier SLC40A1, respectively. For Duroc (contrast (ii)), the clustering of boars based on their gene expression data did not reflect their difference in sperm hyperactivity phenotypes. No results were therefore obtained for this breed. A case-control analysis of variants identified in the Landrace RNA sequencing data showed that SNPs in NEU3, CHRDL2 and HMCN1 might be important for sperm hyperactivity.ConclusionsDifferentially expressed genes were identified in Landrace boars with high and low levels of sperm hyperactivity at the day of ejaculate collection and high and low change in hyperactivity after 96 h of sperm storage. The results point towards important candidate genes, biochemical pathways and sequence variants underlying sperm hyperactivity in pigs.

Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen.

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

Pengaruh Lingkungan dan Nilai Ripitabilitas pada Kualitas Semen Sapi Peranakan Ongole Jantan

The aims of this study were to analyze the effect of environmental factors (age of bull, season of collection and frequency of ejaculation) and estimate the repeatability on semen quality of ongole grade bulls. Semen data were collected from Singosari National Artificial Insemination Centre, in Malang District, East Java Province, Indonesia. A total of 1.020 ejaculates collected from three Ongole grade cattle from 2012-2016 were analyzed. Data were analyzed by restricted maximum likelihood (REML) method using the mixed model including the random effect of bull and the fixed effects of age of bull, season of collection, and ejaculate number. The results showed that age of bull and ejaculate number significantly affected all studied variables (P<0.01). Season only affected to sperm motility (P<0.01). Repeatability estimates of semen volume, sperm concentration, total number of sperm and sperm motility were 0.48; 0.42; 0.28; and 0.01, respectively. It was concluded that the age of the bull and frequency of ejaculation significantly affected semen quality of Ongole grade cattle. Repeatabilities for semen quality were low, moderate and high.

Dependence of physical characteristics of ejaculate of Polish Landrace boars on the time interval between successive ejaculate collections

The aim of the research was to determine the dependence of physical properties of boar ejaculates on the time interval between successive ejaculate collections. The material for the study comprised 1954 ejaculates collected manually from boars used for artificial insemination. For all ejaculates, the ejaculate volume, sperm concentration, percentage of motile spermatozoa, and number of spermatozoa in the ejaculate were determined, and the number of insemination doses per ejaculate was calculated. The boars’ suitability for artificial insemination was evaluated taking into account the effect of the time passed between ejaculate collections. The ejaculate volume and the concentration and number of sperm in the ejaculate were shown to increase with the time elapsed since the previous ejaculate. The number of insemination doses obtained per ejaculate increased as well. Ejaculates with a high sperm count were obtained when the ejaculation frequency was no more than every 4 days. The percentage of spermatozoa with progressive motility increased with the time elapsed since the collection of the previous ejaculate, but only up to 4 days. Further decreases in ejaculation frequency no longer resulted in increased sperm motility.