Semen Doses Research Articles

Effect of Thawing Procedure and Thermo-Resistance Test on Sperm Motility and Kinematics Patterns in Two Bovine Breeds.

This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher (p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values (p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value (p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease (p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

PSVII-28 Unraveling the mechanisms governing boar semen quality during chilled storage

Abstract Artificial insemination (AI) appears as a core of reproductive management in the hog industry. AI involves the use of chilled semen doses meeting routine thresholds for motility (≥ 70%) and morphology (≥ 80%) at collection. However, semen doses meeting these standards exhibit a gradual decline in sperm quality, with likely divergent profiles during preservation that impact fertility outcomes. Hence, this study aims to unravel the sperm quality dynamics of boar semen doses during 7-d chilled storage at 17ºC. Semen doses (n = 25) from proven-fertile Duroc boars were provided by a commercial hog farm (Prestage Farm, MS) and transported to the laboratory within 30 min of collection. Each semen sample was aliquoted and analyzed on the day of collection (d 0) and after 7 d of storage (d 7) at 17°C. Sperm total motility (TM), progressive motility (PM), and normal morphology (NM) were assessed using a computer-assisted sperm analyzer (CASA; CEROS II, IMV Biotechnologies). Apoptosis (Annexin V/7-AAD), viability (Propidium iodide – PI), and intracellular reactive oxygen species (ROS; DCFH-DA) were evaluated using flow cytometry. Total antioxidant activity (TAOC) and lipid peroxidation (malondialdehyde; MDA) were assessed using commercial kits (Cayman Chemical). Data analysis was conducted using parametric statistics (SPSS), with P &amp;lt; 0.05 considered significant. Results were presented as mean ± SEM. Data analysis confirmed the significant decrease of CASA parameters, particularly TM from d 0 to d 7 (78.87 ± 1.37 vs. 73.19 ± 1.98%), with extreme variance on d 7 allowing to discriminate high motility (HM, n = 6) vs low motility (LM, n = 6) semen doses. HM doses evidenced significantly greater (P &amp;lt; 0.05) TM (83.73 ± 1.17%), PM (36.07 ± 1.11%), and NM (89.45 ± 2.30%) compared with LM doses (TM: 59.40 ± 3.35%, PM: 16.57 ± 3.25%, NM: 79.93 ± 2.61%) on d 7. Unlike HM samples, which were comparable between d 0 and d 7, LM doses displayed significantly greater proportions of apoptotic and viable cells, and increased intracellular ROS levels on d 0. Likewise, LM samples revealed significantly greater (P &amp;lt; 0.05) proportions of apoptotic cells (3.69 ± 1.66%), dead cells (38.41 ± 3.71%), and cells with intracellular ROS (36.21 ± 3.62%) than HM samples (apoptotic cells: 0.03 ± 0.02%, dead cells: 4.73 ± 4.65%, ROS: 5.34 ± 4.91%) on d 0; however, only the proportions of dead cells displayed significant increase in LM on d 7. MDA concentrations were comparable between HM and LM samples on d 0. However, on d 7, LM samples exhibited significantly greater MDA concentrations than HM samples. In contrast, TAOC was significantly greater in HM samples (2.53 ± 0.14 mM) compared with LM samples (2.17 ± 0.07 mM) on d 0. In conclusion, semen samples with similar gross characteristics at collection exhibited differential responses during storage. Their initial redox potential and apoptosis-like changes may contribute to their varying ability to withstand prolonged storage. Work supported by USDA-ARS grant# 6066-31000-015-00D.

Buserelin Acetate Added to Boar Semen Enhances Litter Size in Gilts in Tropical Environments.

The use of exogenous hormones has long been of interest for improving reproductive performance in swine production. Enhancing litter size directly impacts the economic efficiency of pig production. Various strategies, including nutritional, genetic, and hormonal approaches, have been explored with varying degrees of success. Administering a gonadotropin-releasing hormone (GnRH) agonist, such as buserelin, at the onset of estrus can induce ovulation and reduce the variation in ovulation timing among sows. This study assessed the impact of GnRH agonist supplementation in boar semen doses on the litter size of inseminated gilts. The research was conducted on a commercial swine herd in northern Thailand. A total of 231 Landrace × Yorkshire crossbred gilts, aged 224.5 ± 16.2 days at the onset of estrus synchronization, participated in the experiment. The gilts' estrus was synchronized with oral altrenogest supplementation at a dosage of 20 mg/day for 18 days. After exhibiting standing estrus, the gilts were randomly divided into three groups. Control group: gilts were inseminated at 0 and 12 h post standing estrus onset with a conventional semen dose (n = 94). Treatment 1: similar to the control group, but with an added 5 µg (1.25 mL) of buserelin acetate to the boar semen dose during the first insemination (n = 71). Treatment 2: similar to the control group, but with 10 µg (2.5 mL) of buserelin acetate added to the boar semen dose during the first insemination (n = 66). All gilts were inseminated twice during their standing estrus using the intrauterine artificial insemination method. Each semen dose contained 3.0 × 109 motile sperm in 80 mL. The farrowing rate averaged 78.8% and did not significantly differ between the groups (p = 0.141). The total number of piglets born per litter in the treatment 2 group was greater than in the control group (14.0 ± 0.3 vs. 13.2 ± 0.3, respectively, p = 0.049), but was not significantly different from the treatment 1 group (13.3 ± 0.3, p = 0.154). Similarly, the number of live-born piglets in the treatment 2 group was greater than in the control and treatment 1 groups (13.2 ± 0.4 vs. 12.3 ± 0.3 and 12.0 ± 0.4, respectively, p < 0.05). Moreover, the live-born piglets' litter birth weight in the treatment 2 group was greater than in the control group (17.0 ± 0.4 vs. 15.6 ± 0.3 kg, respectively, p = 0.008) and the treatment 1 group (15.7 ± 0.4 kg, p = 0.025). In conclusion, adding a GnRH agonist to boar semen appears to enhance the litter size of gilts. Further research should focus on understanding the underlying mechanisms and determining the optimal dose and timing for GnRH agonist supplementation.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration.

Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen.

Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Effect of Antimicrobial Peptide BiF2_5K7K on Contaminated Bacteria Isolated from Boar Semen and Semen Qualities during Preservation and Subsequent Fertility Test on Pig Farm.

The purpose of this study was to determine the impact of an antimicrobial peptide, BiF2_5K7K, on semen quality and bacterial contamination in boar semen doses used for artificial insemination. A key factor affecting semen quality and farm production is bacterial contamination in semen doses. Using antibiotics in a semen extender seems to be the best solution for minimizing bacterial growth during semen preservation. However, concern regarding antibiotic-resistant microorganisms has grown globally. As a result, antimicrobial peptides have emerged as interesting alternative antimicrobial agents to replace the current antibiotics used in semen extenders. BiF2_5K7K is an antimicrobial peptide that can inhibit Gram-negative and Gram-positive bacteria isolated from boar semen and sow vaginal discharge. In this study, ten fresh boar semen samples were collected and diluted with one of two types of semen extender: with (positive control) or without (negative control) an antibiotic (i.e., gentamicin). The semen extender without an antibiotic contained antimicrobial peptide BiF2_5K7K at different concentrations (15.625, 31.25, 62.5, and 125 µg/mL). The samples were stored at 18 °C until use. Semen quality parameters were assessed on days 0, 1, 3, and 5, and the total bacterial count was also evaluated at 0, 24, 36, 48, and 72 h after storage. A fertility test on a pig farm was also performed via sow insemination with a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. No significant difference was found in terms of semen quality on days 0 or 1. On days 3 and 5, the total motility, progressive motility, and viability remained normal in the 15.625 and 31.25 µg/mL groups. However, the sperm parameters decreased starting on day 3 for the 125 µg/mL group and on day 5 for the 62.5 µg/mL group. For total bacterial count at 0, 24, 36, 48, and 72 h, the lowest bacterial count was found in the positive control group, and the highest bacterial count was found in the negative control group compared with the other groups. Comparing antimicrobial peptide groups from 0 to 48 h, the lowest bacterial count was found in the 125 µg/mL group, and the highest bacterial count was found in the 15.625 µg/mL group. For the fertility test, artificial insemination was conducted by using a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. The results showed a superior pregnancy rate, farrowing rate, and total number of piglets born compared with artificial insemination conducted using a commercial extender plus antibiotic. In conclusion, BiF2_5K7K can inhibit bacterial growth in extended boar semen for 24 h, and thereafter, the bacterial count slightly increases. However, the increase in the number of bacterial counts from days 0 to 3 had no negative effect on sperm quality in the positive control, 15.625, or 31.25 µg/mL groups. This indicates that BiF2_5K7K might be an antimicrobial peptide candidate with potential for use as an alternative antimicrobial agent to replace the conventional antibiotic used in boar semen extenders.

Sow reproductive performance following artificial insemination with semen doses processed using Single Layer Centrifugation without antibiotics in the tropics

Single Layer Centrifugation (SLC) through a low density colloid offers an alternative solution to antibiotic use in boar semen extenders, with lower costs compared to high density colloids. The aim of this study was to explore the reproductive performance of sows when using SLC-prepared semen doses without antibiotics, employing low density Porcicoll to prepare semen doses for artificial insemination in a commercial swine herd in Thailand. Ejaculates were divided into two equal parts to create insemination doses, with each dose containing 3000 × 106 sperm/80 ml for intra-uterine insemination in individual sows. The sows were inseminated twice, with the interval between the two inseminations ranging from 8 to 16 h. The CONTROL group consisted of 206 semen doses treated with antibiotics, prepared for insemination in 103 sows, while the SLC group comprised 194 SLC-prepared semen doses without antibiotics for inseminating 97 sows. Fertility and fecundity traits, including non-return rate, conception rate, farrowing rate, and litter traits (i.e., the total number of piglets born per litter, number of piglets born alive per litter, number of stillborn piglets, and number of mummified fetuses), were compared between groups. Furthermore, data on piglet characteristics, including live-born and stillborn piglets (i.e., the prevalence of stillbirth (yes, no), birth weight, crown-rump length, body mass index (BMI), and ponderal index (PI)), were determined. No significant differences in non-return rate (75.7 % vs. 77.3 %), conception rate (73.8 % vs. 73.2 %), and farrowing rate (71.8 % vs. 73.2 %) were observed between the CONTROL and SLC groups, respectively (P > 0.05). Nevertheless, the total number of piglets born per litter in the SLC group was higher than in the CONTROL group (14.6 ± 0.9 vs. 12.3 ± 0.6, respectively, P = 0.049). Interestingly, the prevalence of stillbirth in the SLC group was lower than in the CONTROL group (6.2 % vs. 11.6 %, respectively, P < 0.001). Moreover, the newborn piglets in the SLC group exhibited higher birth weight and BMI compared to those in the CONTROL group (1.36 ± 0.03 vs. 1.26 ± 0.02 kg, P = 0.005, and 18.3 ± 0.3 vs. 17.3 ± 0.2 kg/m2, P = 0.003). In conclusion, employing sperm doses after SLC through a low density colloid in artificial insemination within a commercial breeding operation did not have a detrimental impact on either fertility or fecundity traits but showed potential benefits in increasing the total number of piglets born per litter. Moreover, improvements were observed in the birth weight and body indexes of piglets, and the percentage of stillbirths was reduced. Our findings introduce new possibilities for antibiotic alternatives in semen extenders to reduce the risk of antimicrobial resistance in the swine industry. Additionally, they provide compelling reproductive outcomes supporting the integration of SLC-prepared semen doses into artificial insemination practices.

Update of the cooling protocol for antibiotic-free storage of boar semen at 5°C improves sperm quality and maintains low bacterial counts.

Preserving boar semen at 5°C instead of the conventional storage temperature of 17°C would enable a reduction of antibiotic use in pig insemination. To protect the chilling-sensitive boar spermatozoa, holding the extended semen at a higher temperature before cooling could be beneficial and facilitate the implementation of the innovative preservation concept in practice, provided that bacterial growth is kept at a low level. The aim of this study was to introduce a holding time (HT) at 17°C before cooling and to examine the effect on sperm quality and bacterial growth compared to the original cooling protocol for antibiotic-free 5°C semen storage. A series of experiments with semen doses from eight boars extended in Androstar® Premium without conventional antibiotics revealed that sperm kinematics and the integrity of sperm plasma membranes and acrosomes were improved with HT between 16 and 24 h followed by delayed cooling with 0.04°C/min when compared to the original protocol for semen preservation at 5°C (p < 0.05). Both a shorter HT of 6 h and a faster cooling rate of 0.07°C/min reduced sperm quality (p < 0.05). The HT for 24 h did not compromise the inhibitory effect on bacterial growth during long-term semen storage at 5°C, not even in semen doses spiked with Serratia marcescens. In conclusion, semen storage at 5°C with the modified cooling protocol improved sperm quality and is antimicrobially efficient. It thus presents a ready-to-use tool for a reduction or replacement of antibiotics in pig insemination.

Effect of repeated semen ejaculation on sperm quality and selected biochemical markers of canine semen

The aim of this study was to evaluate the quality parameters and selected biochemical markers of canine semen sampled at 24-h intervals over a period of 5 days, preceded by 6 months of sexual abstinence. Full ejaculates were obtained from 6 dogs. Ejaculate volume and total sperm counts in the ejaculate decreased gradually on successive sampling days. The percentage of total motile spermatozoa (TMOT), percentage of progressively motile spermatozoa (PMOT), sperm plasma membrane integrity (SPMI), and sperm mitochondrial membrane potential (MMP) increased on successive days of sampling. In addition, ATP content increased in spermatozoa. Total protein content (TPC) and the activity of aspartate aminotransferase (AAT), alkaline phosphatase (AP), and acid phosphatase (AcP) decreased in seminal plasma. Repeated ejaculation over a period of 5 days induced changes in the qualitative and quantitative parameters of canine semen. A decrease in the values of some biochemical markers of semen, secreted by the epididymis and the prostate gland, could point to disturbances in the secretory activity of these organs. Canine semen sampled after prolonged sexual abstinence is generally characterized by less desirable quality parameters, and this observation should be taken into consideration when semen is collected for artificial insemination or preservation. Semen quality can be significantly improved by repeating the sampling procedure after 24 hours. One the other hand, repeated sampling on successive days can significantly decrease total sperm counts in the ejaculate. As a result, a sufficient number of semen doses for artificial insemination may not be obtained from a single ejaculate.

Evaluation of predictive ability of linear type gonadal traits on reproductive capacity of breeding dairy bulls.

Dimensions of linear type traits facilitate selection of livestock for breeding and rearing. To date, use of linear type traits for selection of breeding bulls is highly concentric to scrotal circumference (SC), with probable overlook to other important traits. Present study reported the importance of various gonadal linear type traits on spermatozoa production, age-related changes in gonadal linear type traits of bulls and predictive ability of these traits on bulls' reproductive potentials. Among all gonadal traits, testicular density (TD), scrotal volume (SV), paired testicular weight (PWT) and SC were found most important predictor variables in order, which can discriminate between good/poor breeding bulls, that is, produced frozen semen doses (FSD) or not. Dimensions of gonadal traits increased significantly up to 36 months age and thereafter, development became slow and negligible. In contrast, TD decreased by 30%, 51%, 64%, 68% and 71% at 12, 24, 36, 48 and >49 months age, respectively, from its base value at 6 months. Bulls of lower TD (≤0.88 g/cm3) had significantly higher ejaculate volume (+9%), sperm motility, sperm concentration (+100 million/mL) and sperm output (+26%)/ejaculate as compared to bulls of higher TD (>0.88 g/cm3). Discriminant function was developed using TD, SV, PWT and SC to identify bulls of superior reproductive potentials. It was concluded that among the investigated traits, TD was the strongest to discriminate between FSD and Non-FSD bulls. Therefore, our findings suggested that TD could be more potential trait than SC for dairy bulls' breeding soundness evaluation and assessment of reproductive ability.

Vibration Emissions Reduce Boar Sperm Quality via Disrupting Its Metabolism.

Artificial insemination (AI) with liquid-preserved semen has recently become common in pig breeding. The semen doses are produced in a centralized manner at the boar stud and then subsequently distributed and transported to sow farms. However, vibration emissions during transportation by logistic vehicles may adversely affect the quality of boar sperm. Therefore, this study aimed to explore the impact of vibration-induced emissions on sperm quality and function under simulated transportation conditions. Each time, ejaculates from all 15 boars were collected and then pooled together to minimize individual variations, and the sample was split using an extender for dilution. Different rotational speeds (0 rpm, 80 rpm, 140 rpm, 200 rpm) were utilized to simulate varying intensities of vibration exposure using an orbital shaker, considering different transportation times (0 h, 3 h, and 6 h). Subsequently, evaluations were conducted regarding sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial function, adenosine triphosphate (ATP) levels, mitochondrial reactive oxygen species (ROS) levels, pH, glycolytic pathway enzyme activities, and capacitation following exposure to vibration emissions. Both vibration time and intensity impact sperm motility, plasma membrane integrity, and acrosomal integrity. Vibration exposure significantly reduced sperm ATP levels, mitochondrial membrane potential, and the levels of mitochondria-encoded proteins (MT-ND1, MT-ND6) (p < 0.05). After vibration emission treatment, the pH value and mitochondrial ROS levels significantly increased (p < 0.05). Inhibition of sperm glycolysis was observed, with reduced activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), along with decreased lactate levels (p < 0.05). Additionally, sperm tyrosine phosphorylation levels were significantly reduced by vibration emissions compared to the control group (p < 0.05). After the vibration emission treatment, the number of sperm bound to each square millimeter of oviduct explants decreased significantly compared to the control group (p < 0.05). Similarly, compared to the control group, using semen subjected to vibration stress for AI results in significantly reduced pregnancy rates, total born litter size, live-born litter size, and healthy born litter size (p < 0.05).

PSIII-9 Field fertility following the practice of post-cervical artificial insemination (PCAI) at multiple North American swine units

Abstract On a global scale, greater than 95% of sows are bred using artificial insemination (AI). As with most technologies, efficiencies improve over time and in the case of swine AI the industry is moving towards fewer sperm per dose in conjunction with a post-cervical deposition (PCAI). Nevertheless, little information is available stating the fertility of farms actively using PCAI. The objective of this research is to provide a retrospective report on the field fertility of multiple farms in North America utilizing PCAI. As a reference, information is provided for contemporary farms utilizing conventional AI (intra-cervical deposition in conjunction with a greater total sperm count per dose). Semen doses produced for PCAI (1.5 billion total cells in 40 mL volume) sourced from one boar stud located in Tennessee and are packaged as pooled semen in NUTRIXcell + extender. Semen was held overnight at 18°C and delivered to seven farms the next day. Semen doses produced for conventional AI (CONAI) also sourced from a single boar stud located in Michigan; packaged as pooled semen at 2.75 billion total cells in a 70 mL volume using the NUTRIXcell + extender and shipped to six farms the same day as semen production. Both PCAI and CONAI were used on 100% of gilts and multiparous sows in respective systems. Field fertility, reported as farrow rate (FAR) and mean total born (TB), is reported below for both PCAI (Table 1) and CONAI (Table 2) following use per individual farms during January 2022 to September 2023. Results suggest that PCAI can maintain acceptable levels of fertility and should enable cost savings in conjunction with increased dissemination of superior genetics.

Comparative Study on the Use of Artificial Insemination in Sows in Three Production Units in the Region of Tlachichuca Puebla, México

The main objective of this study was to make a comparison of the use of artificial insemination in sows from three production units belonging to the Tlachichuca-Puebla region, Mexico. These pork production units belonged to the communities of José María Morelos, San Francisco Independencia and Tlachichuca, Puebla respectively. 20 sows were used, 15 of which were primal and 5 multiparous, which were inseminated over a period of 10 months. 20 doses of semen were acquired from the Coyametl Genetic Transfer Center, located in the City of Acatzingo, Puebla. The information obtained from the project was concentrated in the different stages of the study, and stored in an Excel file; the information was processed through the SPSS 10 package for Windows, applying descriptive statistics. 75% of Primal or Nulliparous sows were found which were artificially inseminated and 25% corresponded to Multiparous; 7% of the producers expressed no interest in planning but rather a greater number of kilos when selling, 15% responded interest in the number of offspring and fewer deaths at birth with better feeding of the bellies before and after the births, 78% mentioned the importance of planning a procedure to improve their productivity and reproductive life in their pork production units (PPUs). Regarding litter size, results were obtained through AI in the sows of the 3 UPPs (José María Morelos, San Francisco Independencia and Tlachichuca), a total of piglets in the primals of 163 of the 15 inseminated sows, with an average of 10.8 piglets/sow; In the case of multiparous sows, there were 58 piglets in total with an average of 11.6 piglets/sow. In conclusion, comparative studies are useful for analysis in pork production units, since they are pillars in decision making; as it was, in the reproductive indicators where AI was applied, giving objectivity and greater certainty as was the case in the three communities of the Tlachichuca region, Puebla, Mexico.

Physiological features of semen resistance of stallions to cooling with different antigenic profile of erythrocytes

The article shows the physiological features of the survival ability of stallion semen at a temperature of 2 to 5°C with different antigenic features according to blood group systems. It was shown that in the presence of the antigens cgm/ceg, ad/dk, cgm/dk, cgm/d, dg/di on D system erythrocytes, the semen survival ability at 2 to 5°C was observed for a maximum of 50 hours. The carriers of the antigens cegm/cgm, ad/de, cgm/cgm, аd/bcm, bcm/dg, ad/d, cegm/dg, bcm/cgm, bcm/de, cegm/d, de/cgm, cegm/dk, dk/d, de/d, de/dk, cgm/dg, dk/de antigens have obtained a medium sperm resistance to cooling with a semen survival time of 50 to 75 hours. Stallions with the antigens bcm/d, bcm/dk, ad/cgm, dg/dk, cgm/de, dg/cgm, dk/dk of the D blood group showed a high semen resistance to cooling at 2 to 5°C in the form of a survival time of more than 75 hours. When the experimental stallions inherited the a/- antigen of the K system, the semen survival ability was increased by 7.24 h (P &lt; 0.05) and the absolute survival ability by 24.18 standard units (P &lt; 0.05) compared to the absence of this system antigen. The absence of -/- erythrocytic antigens of the C blood group is associated with an increase (P &lt; 0.05) of 10.21 h in the survival ability and 30.29 standard units in the absolute semen survival ability compared to ejaculates from stallions with the a/- antigen of this erythrocytic system. The absence of the -/- antigen of the A system in stallions and the presence of the а/- antigen are followed by an increase in ejaculate resistance to cooling by 14 h (Р &lt; 0,05) as compared to the control. At the same time, the absolute semen survival ability is 38.69 and 45.94 standard units higher than the control values (P &lt; 0.01). Inheritance by stallions of the ad/- antigen by stallions was followed by a higher semen resistance to cooling as compared to the control by only 9.63 h (Р &lt; 0.05) and a lower resistance compared to а/- and -/- by 5 h. The practical application of the data obtained is that it becomes possible to predict the shelf life of freshly dissolved cooled semen before artificial insemination, which is especially important for long transport of semen doses.

108 Discriminating highly resilient spermatozoa during long-term chilled storage

Abstract Artificial insemination (AI) is the most important assisted reproductive technique devised to enhance the genetic potential of animals by enabling widespread utilization of elite males. In the swine industry, AI with extended semen is highly effective, with several extenders developed for long-term storage. Regardless, the gradual decline of sperm quality during storage remains a major concern for rapid genetic dissemination. We hypothesized that existing sire-to-sire variations could reflect on sperm survival during prolonged storage. Therefore, this study examined boar sperm motility and morphology profiles to determine potential disparities during long-term storage within a commercial extender. Freshly collected Duroc boar semen samples (n = 58 boars) were extended at a commercial boar stud (Prestage Farms, MS), and sample doses (n = 29) were appropriately transported to the laboratory for experiments. Semen samples were aliquoted in tubes (filled to the top) and immediately stored in an incubator set at 17°C for up to 10 d post-collection. Daily, samples were assessed for total motility (TM), progressive motility (PM), velocities [average path velocity (VAP), curvilinear velocity (VCL), and straight-line velocity (VSL)], and normal morphology (NM) using the computer-assisted sperm analyzer (CASA, CEROS II). The deviations of TM means were used for survival discrimination between so-called “good” and “bad” semen. Data were log-transformed and analyzed (repeated ANOVA and Bonferroni adjustment) to determine the effect of time during storage. Results are expressed as mean ± SEM and P &amp;lt; 0.05 indicates significant differences. As expected, all data demonstrated a consistent and significant decline over time (P &amp;lt; 0.05), from day-0 to day-10 (TM: 78.2 ± 1.0% to 42.8 ± 4.3%; PM: 46.1 ± 1.8% to 16.2 ±2.3%, and NM: 85.1 ± 1.0% to 76.1 ± 1.9%). Notably, velocity parameters (VAP, VSL, and VCL) remained relatively stable during the storage period, while the distal droplets contributed the most to morphological defects (54.1±1.5% vs. 28.3±1.5%, 18.3±0.5%, and 2.4±0.2% for the proximal droplets, bent and coiled tailed, respectively; P&amp;lt; 0.05). Survival discrimination indicated the highest deviations from the overall averages on day-7.Consequently, good (n = 8) and bad (n = 7) semen samples had significantly different (P &amp;lt; 0.05) in TM (80.2 ± 1.4% and 21.7 ± 4.6%, respectively), PM (39.7 ± 2.7% and 4.7 ± 1.7%, respectively), and NM (87.9 ± 1.9 and 66.4 ± 2.6%, respectively).Similarly, the velocity parameters were significantly different (P &amp;lt; 0.05) between both sample groups, including VAP (74.9 ± 2.6 µm/s vs. 40.4 ± 8.7 µm/s), VCL (135.2 ± 6.0µm/s vs. 81.9 ± 17.3 µm/s), and VSL (59.0 ± 1.8 µm/s vs. 31.3 ± 6.8 µm/s). These findings indicate that specific subsets of boar semen may be well-suited for survival during long-term chilled preservation. The development of tools for early discrimination of semen doses, immediately following collection, could significantly enhance their management and facilitate appropriate shipments to remote areas or during adverse weather conditions. Ongoing studies are dedicated to unraveling the cellular mechanisms underlying the resilience of such spermatozoa. - Research funded by USDA-ARS, grant#58-6066-3-038.

Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The ‘sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.

Easy of calving cows for dairy pro- ductivity

Data are presented on the dependence of the ease of calving on the duration of the dry period, gender, the number of gestated fetuses and its influence on the frequency of occurrence of specific pathologies of childbirth and the postpartum period and the frequency of insemination in dairy cows of productivity in the conditions of Sibirskaya Niva LLC, Novosibirsk region. More often, childbirth proceeded normally (easy), without outside intervention - 91.36 %, mild and moderate pathology was observed in 3.35 and 4.41 % of cows, and in rare cases, calving was accompanied by severe pathology - 0.87 %. The severity of the birth process increased with a decrease in the duration of the dry period. Thus, in animals with easy labour, the dry period was 54.73 ± 0.27 days; in animals with severe pathology, it was three days less - 51.74 ± 1.33 days. In cows with mild labour, the birth of one calf was more often observed; the number of twins did not exceed 8.95 %. The severity of labour was accompanied by an increase in the number of twins; in the group of animals with arduous labour, it reached 84.21 %. With a problematic labour process, the frequency of stillbirths in groups increases by 4.41 %. Animals with difficult births have an increased risk of obstetric and gynaecological pathology. Thus, in cows with a severe course of the birth process, in 26.32 % of cases, pathology of the third stage of labour was observed, accompanied by retention of the placenta. In the postpartum period, postpartum paresis was often recorded - 42.11 % and various types of metritis - 21.05 %. In contrast, with a mild course of labour, all the pathologies we studied were recorded in aggregate only in 29.35 % of cows. In animals with arduous labour, 1.05 more doses of semen were spent on fertile insemination than in animals with normal labour.

Sperm quality and in vitro fertilizing ability of boar spermatozoa stored at 4 °C versus conventional storage for 1 week.

Since boar spermatozoa show a marked deterioration in sperm quality when cooled, insemination doses are usually stored at 16-18 °C. However, maintaining this temperature during transport of semen doses is challenging, particularly during the summer months. An alternative could be to store the doses at 4 °C if cold-shock to the sperm could be prevented. The objective of this study was to evaluate boar sperm quality and fertility in in vitro fertilization after storage in AndroStar Premium at 4 °C for 1 week. Insemination doses (n = 9) in AndroStar Premium from a commercial boar semen collection station were transported to the laboratory at approximately 20 °C. At the laboratory, sperm quality evaluation and was preformed and each dose was split; half of each ejaculate was stored in a climate-controlled box at 16-18 °C, the other was slowly cooled to 4 °C. Both samples were stored for 1 week before further sperm quality evaluation and in vitro fertilization (IVF) were performed. Mean values were tested using generalized linear regression, with treatment and boar as fixed factors; p ≤ 0.05 was considered significant. Sperm membrane integrity (mean ± sem: 91 ± 0.05 and 83 ± 0.09% for 16 and 4 °C, respectively) and superoxide production (6.79 ± 2.37 and 13.54 ± 6.23% for 16 and 4 °C, respectively), were different between treatments. The DNA fragmentation index was lower in cold-stored samples than in conventionally stored samples (3.74 ± 2.25 and 7.40 ± 3.36% for 4 and 16 °C, respectively). The numbers of oocytes developing to blastocyst on Day 6 (mean ± sd: 9.0 ± 8.0 and 6.0 ± 5.0%, for storage at 16 and 4 °C, respectively) were not different between treatments. Therefore, storage of boar semen doses in AndroStar Premium at 4 °C for up to 7 days would be a viable alternative to current praxis.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Laboratory testing and on-site storage are successful at mitigating the risk of release of foot-and-mouth disease virus via production of bull semen in the USA.

Thousands of frozen bovine semen doses are produced daily in the US for domestic use. An incursion of foot-and-mouth disease (FMD) in the country would pose strong challenges to the movements of animals and animal products between premises. Secure supply plans require an estimation of the risk associated with target commodities and the effectiveness of mitigation measures. This study presents the results of a quantitative assessment of the risk of release of FMD virus from five of the largest commercial bull studs in the US via contaminated frozen processed semen. The methodology from a previous study was adapted to better fit the US production system and includes more recent data. Two models were combined, a deterministic compartmental model of FMD transmission and a stochastic risk assessment model. The compartmental model simulated an FMD outbreak within a collection facility, following the introduction of a latent-infected bull. The risk of release was defined as the annual likelihood of releasing at least one frozen semen batch, defined as the total amount of semen collected from a single bull on a given collection day, containing viable FMD virus. A scenario tree was built using nine steps leading from the collection to the release of a contaminated batch from a given facility. The first step, the annual probability of an FMD outbreak in a given facility, was modeled using an empirical distribution fitted to incidence data predicted by five models published between 2012 and 2022. An extra step was added to the previously published risk pathway, to account for routine serological or virological surveillance within facilities. The results showed that the mitigation measures included in the assessment were effective at reducing the risk of release. The median annual risk of release from the five facilities was estimated at less than 2 in 10 billion (1.5 x 10-10) in the scenario including a 30-day storage, routine genome detection assays performed every two weeks and RT-PCR testing of the semen. In this scenario, there was a 95% chance that the risk of release would be lower than 0.00041. This work provides strong support to the industry for improving their response plans to an incursion of FMD virus in the US.