Artificial Insemination Doses Research Articles

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.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints.The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Bicarbonate and BSA increase the capacitation pattern and acrosomal exocytosis in boar sperm after 120 min of incubation.

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the invivo situation, there is still a lack of standardization invitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on invitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence invitro sperm capacitation in this species.

56 Biomarker-Based High Throughput Sperm Phenotyping: the Good, the Bad and the Ugly

Abstract Research in our laboratory addresses the unmet need for developing high throughput phenotyping for collecting comprehensive phenotypes of production, reproduction and fitness traits, a major emphasis area of the USDA Blueprint for Animal Genome Research 2022-2027. Our short- and long-term goal within this emphasis area is to identify specific genomic determinants of aberrant sperm quality and integrity that impart unique morphometric and optical properties on functionally defective spermatozoa that can be captured in next generation flow cytometry at a high speed/throughput, and without the need for costly, time-consuming sample processing. We identified a large set of deleterious gene variants associated with specific sperm defects in the genomes of 85 genetically valuable bulls with suboptimal artificial insemination (AI) fertility. Ongoing efforts focus on developing a second generation, label-free sperm phenotyping pipeline, eliminating variance and costs associated with the conventional, probe-based approaches, based on the specific genomic determinants of aberrant sperm quality resulting in unique morphometric and optical properties of spermatozoa, captured by the next-generation, brightfield image-based flow cytometry (IBFC) and translatable to high-precision phenotyping of heritable production traits. First step is to phenotype and validate rare deleterious polymorphisms associated with aberrant sperm head and tail morphologies in aforementioned cohort of 85 genome-sequenced bulls grouped based on prevailing sperm defect phenotype. Focus is on sperm defects predicted to be heritable and non-compensable by increasing sperm number per AI dose, such as knobbed acrosomes (sperm head caps), nuclear vacuoles and aplastic defects of sperm mitochondrial sheath (the major source of energy to fuel sperm movement). Other potentially heritable defects of interest may not be manifested by aberrant sperm morphologies, and only detectable at subcellular/molecular level by applying biomarkers validated by past and ongoing genome-to-phenotype studies. In the second phase, a label free sperm phenotyping pipeline for bull fertility testing will be developed, translatable to somatic cell phenotyping for other heritable traits by using the dedicated FeatureFinder IFBC software integrated with our machine learning pipeline, followed by validation assessing sperm structural integrity, “gentle” live cell sorting and metabolomics. This approach literary puts artificial intelligence in artificial insemination (AI in AI approach). Altogether, such genome-to-phenome efforts align the livestock genomic selection with state-of-the-art animal andrology, veterinary diagnostics and precision breeding, supporting the improvement of livestock industry efficiency through global approaches integrating production, reproduction, nutrition, health, and welfare.

Screening for atypical porcine pestivirus in Swedish boar semen used for artificial insemination and a characterisation of the seminal RNA microbiome including the virome

BackgroundThis study aimed to characterise the RNA microbiome, including the virome of extended semen from Swedish breeding boars, with particular focus on Atypical porcine pestivirus (APPV). This neurotropic virus, associated with congenital tremor type A-II in piglets, was recently demonstrated to induce the disease through insemination with semen from infected boars.ResultsFrom 124 Artificial Insemination (AI) doses from Swedish breeding boars, APPV was detected in one dose in addition to a sparse seminal RNA virome, characterised by retroviruses, phages, and some fecal-associated contaminants. The detected seminal microbiome was large and characterized by Gram-negative bacteria from the phylum Proteobacteria, mainly consisting of apathogenic or opportunistic bacteria. The proportion of bacteria with a pathogenic potential was low, and no antimicrobial resistance genes (ARGs) were detected in the datasets.ConclusionOverall, the results indicate a good health status among Swedish breeding boars. The detection of APPV in semen raises the question of whether routine screening for APPV in breeding boars should be instigated.

Rotation of liquid-preserved artificial insemination doses on roller benches affects sperm quality during storage in stallions.

Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of diluted stallion AI portions on sperm quality. Ejaculates (n = 15) were collected at a German AI centre and diluted with the two extenders EquiPlus and Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL. Afterwards, samples were placed separately on roller benches at 5°C in the dark, obtaining a rotation frequency of 5 revolutions per minute (rpm) and 36 rpm for four consecutive days following a split-sample design. Both groups were analysed daily in comparison to a control group (0 rpm) with an extended spectrum of spermatological methods including computer-assisted sperm analysis and flow cytometry. Statistical analyses were based on the calculation of generalized linear mixed models for each sperm parameter. The research revealed a decrease in sperm quality parameters of rotated samples compared to non-rotated control groups, visible in total sperm motility (p < .001), decreased thermo-resistance (p < .01) and a drop in pH (p < .001). Interestingly, no differences (p > .05) were detected between rotation frequencies of 5 and 36 rpm. We conclude that the fertilizing capacity of stallion semen was negatively affected by rotation during storage in vitro, irrespective of the rotation frequency. Further studies need to investigate whether field fertility in horses is similarly affected by semen rotation on roller benches in vivo.

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection.

Boar Semen Shipping for Artificial Insemination: Current Status and Analysis of Transport Conditions with a Major Focus on Vibration Emissions.

Simple SummaryLiquid preserved boar semen is a perishable product, and many environmental influences can affect sperm quality during transport from the boar station to the sow farm. Previous studies have shown that vibration emissions have a negative effect on spermatozoa, however, there has been no documentation of the vibrations spermatozoa are exposed to during transport. To answer this question, several breeding companies worldwide were interviewed about their delivery process. As it turns out, environmental influences are only rarely recorded, and vibrations are not monitored at all. To record and to evaluate vibrations that occur during transport, we have developed a measurement system and investigated vibration intensities in standardized road trials. We were able to show that both speed and road surface significantly influence the produced vibrations. Furthermore, the occurrence of the vibrations on a reference delivery from a German boar stud was studied. In the future, it is recommended that in order to avoid negative influences during transport, the transport of boar semen should be permanently monitored.In the modern pig reproduction system, artificial insemination (AI) doses are delivered from AI centers to sow farms via logistics vehicles. In this study, six breeding companies in three countries (Brazil, Germany, and the USA) were interviewed about their delivery process. It was found that there is currently no comprehensive monitoring system for the delivery of semen. The entire process “shipping of boar semen” was documented using Business Process Model and Notation (BPMN). Although it is not currently known which vibrations occur at all, it is suspected that vibration emissions affect the quality of boar semen. For this reason, a prototype of a measuring system was developed to calculate a displacement index (Di), representing vibration intensities. Vibrations were analyzed in standardized road trials (n = 120) on several road types (A: smooth asphalt pavement, B: rough asphalt pavement, C: cobblestone, and D: dirt road) with different speeds (30, 60, 90, 120, and 150 km/h). A two-way ANOVA showed significant differences in mean Di, depending on road surface and speed as well as an interaction of both factors (p < 0.001). A field study on a reference delivery from a German AI center to several sow farms indicated that 33% of the observed roads are in good quality and generate only a few vibrations (Di ≤ 1), while 40% are of a moderate quality with interrupted surfaces (Di = 1–1.5). However, 25% of the roads show markedly increased vibrations (Di ≥ 1.5), as a consequence of bad conditions on cobblestones or unpaved roads. Overall, more attention should be paid to factors affecting sperm quality during transport. In the future, an Internet of Things (IoT) based solution could enable complete monitoring of the entire transport process in real time, which could influence the courier’s driving behavior based on road conditions in order to maintain the quality of the transported AI doses.

High genetic correlation for milk yield across Manech and Latxa dairy sheep from France and Spain

Spanish Latxa and French Manech are dairy sheep breeds that split into Blond (Latxa Cara Rubia, LCR; Manech Tête Rousse, MTR) and Black (Latxa Cara Negra of Navarre, LCN; Manech Tête Noire, MTN) strains. Exchange of genetic material (artificial insemination doses) is becoming more and more frequent across these breeds, within color, to boost both genomic precision using a larger reference population and genetic progress using a larger selection base. This exchange leads to some rams having descendance across both countries. However, additional gains can only be achieved if the selected traits are genetically similar across countries. The objective of this work was to estimate the genetic correlation across breeds for milk yield. We combine across-country, within-color records, pedigree, and marker information. The number of animals with records oscillates from 65,000 (LCN) to 544,000 (MTR), whereas the number of connecting artificial insemination rams (with more than 10 daughters in the other country) is 381 MTR rams in LCR and 58 MTN rams in LCN. Blond strains had a stronger and more extended-in-time connection. The number of genotyped rams goes from 328 (LCN) to 4,901 (MTR). The relatedness of populations was assessed by principal component analysis and Fst coefficients. The genetic correlation was estimated using 2 (one per color) 2-trait models (each country a trait), including all available data (records, pedigree and genotypes), by maximum profile likelihood while fixing other variance components to within-population estimates. Results showed a closer genetic relationship of Blond strains than of Black strains (Fst: 0.01 vs. 0.05, respectively). Genetic correlation estimates for milk yield were 0.70 in both cases. Based on Fst distances, we expected a lower correlation for Black strains than for Blond ones if dominance or epistasis are important. Thus, we attribute the value of this correlation not being close to 1 mostly to genotype-by-environment interaction, including on-farm management and trait modeling. Regardless, the correlation of 0.7 across populations is encouraging for future joint work of Latxa and Manech breeders, including joint genetic evaluations.

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.

Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq.

RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPureTM on porcine sperm. Four boar ejaculates were purified with BoviPureTM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high—although not complete—similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs.

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics.

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1‐hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane‐intact spermatozoa determined by computer‐assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen‐thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.

Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage.

Simple SummaryOne of the problems with the refrigerated storage of spermatozoa in goats is the short shelf life. The study of the effect of the different storage conditions on the different sperm parameters could help to increase sperm survival. In this work, we studied the effect of temperature and sperm concentration during refrigerated storage on sperm motility, mitochondrial membrane potential and DNA fragmentation. We observed that refrigerated storage of semen doses reduced the majority of sperm quality parameters, however, sperm DNA fragmentation was not affected. Storage at 5 °C preserved higher sperm motility than at 17 °C. Moreover, the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa.The use of cooled semen is relatively common in goats. There are a number of advantages of cooled semen doses, including easier handling of artificial insemination (AI) doses, transport, more AI doses per ejaculate, and higher fertility rates in comparison with frozen AI doses. However, cooled semen has a short shelf life. The objective of this study was to examine the effect of temperature and sperm concentration on the in vitro sperm quality during liquid storage for 48 h, including sperm motility and kinetics, response to oxidation, mitochondrial membrane potential (MMP) and DNA fragmentation in goats. Three experiments were performed. In the first, the effects of liquid preservation of semen at different temperatures (5 °C or 17 °C), durations (0, 24 and 48 h) and sperm concentrations (250 × 106 sperm/mL (1:2 dilution rate), 166.7 × 106 sperm/mL (1:3 dilution rate) or 50 × 106 sperm/mL (1:10 dilution rate)) on sperm motility and kinetics were studied. In the second experiment, the effect of temperature, sperm washing and concentration on sperm motility and DNA fragmentation was studied. Finally, the effect of sperm concentration and duration of storage at 5 °C on sperm motility, response to oxidative stress and MMP was examined. We found that refrigerated liquid storage of goat sperm impaired sperm quality, such as motility, MMP and response to oxidation, as storage time increased; however, sperm DNA fragmentation index was not significantly affected. Liquid storage at 5 °C preserved higher total motility than at 17 °C. Moreover, we observed that the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa conserved in milk-based extender in the conditions tested.

Influence of transcervical infusion of seminal plasma on the farrowing rate and litter size in artificially inseminated sows

ABSTRACT Recent studies have focused on the use of seminal plasma to increase sow fertility after classical intracervical artificial insemination (AI). The aim of the present study was to investigate the influence of seminal plasma infusion, prior to the application of conventional AI dose, on the fertility rate in sows. A total of 114 sows were treated with intrauterine infusion of 30ml seminal plasma (SP), while 114 control sows were infused by physiological solution (PS), immediately before the application of conventional AI dose. The experiment was conducted at one commercial pig farm in Serbia, which is comprised of 1,500 sows in the breeding herd. Intrauterine infusion of seminal plasma produced significantly (P&lt;0.05) higher farrowing rate (93.8%) and significantly (P&lt;0.01) more live-born piglets per litter (12.27), compared with the control sows (83.33% farrowing rate and 10.48 piglets). The present results show that intrauterine infusion of seminal plasma can be a useful tool for increasing the fertility rate in artificially inseminated sows, under the conditions of practical intensive pig production.

Physiological effects on rabbit sperm and reproductive response to recombinant rabbit beta nerve growth factor administered by intravaginal route in rabbit does

Beta nerve growth factor (β-NGF) is present in the seminal plasma of some species, including rabbits, acting as an ovulation-inducing factor in camelids. Traditionally, GnRH analogues are used to induce ovulation by intramuscular route when artificial insemination (AI) is performed in rabbit does. A specific rabbit recombinant β-NGF (rrβ-NGF) produced in our laboratory was tested as an alternative method to conventional treatment with GnRH analogues to induce ovulation. In the present work, different concentrations (0, 20, 100 ng/mL and 1, 20 and 100 μg/mL) of rrβ-NGF were added to diluted semen to assess its effect on sperm traits (viability and motility parameters). rrβ-NG was used also, incorporated to the AI dose, to evaluate ovulation response (LH and progesterone plasma concentrations, ovulation rate (OR) and embryo implantation at Day 7) after intravaginal administration. A negative control group stimulated with an empty catheter, and a positive control group inseminated and intramuscularly treated as usual with GnRH were also set up. Results showed that seminal quality was influenced by rrβ-NGF depending on the concentration added, being the highest concentrations tested deleterious for semen. Whereas the highest OR was found in the positive control group (100%), concentrations of 20 ng/mL, 1 μg/mL and 20 μg/mL of rrβ-NGF triggered intermediate OR (30, 60 and 42.9%, respectively), and 100 ng/mL and 100 μg/mL had the lowest OR (20 and 14.3%, respectively). Although LH peak was not observed in the first 2 h after AI in the ovulated females from rrβ-NGF groups, plasma progesterone significantly increased at Day 7, except in those females treated with 20 and 100 μg/mL. Also, 98.4% of ovulated females were pregnant on Day 7. In conclusion, rrβ-NGF added to diluted semen affects seminal quality and provokes ovulation, the development of functional CL and conception by intravaginal route in rabbit does, depending on the concentration added.

Differential seminal plasma proteome signatures of boars with high and low resistance to hypothermic semen preservation at 5°C.

Hypothermic storage at 5°C has been investigated as an alternative to promote the prudent use of antibiotics for boar artificial insemination doses. However, this temperature is challenging for some ejaculates or boars. The present study aimed to identify putative biomarkers for semen resistance to hypothermic storage at 5°C by comparing the seminal plasma proteomes of boars with high and low seminal resistance to preservation at 5°C. From an initial group of 34 boars, 15 were selected based on the following criteria: ejaculate with ≤20% abnormal spermatozoa and at least 70% progressive motility at 120hours of storage at 17°C. Then, based on the response to semen hypothermic storage at 5°C, boars were classified into two categories: high resistance-progressive motility of >75% in the three collections (n=3); and low resistance-progressive motility of <75% in the three collections (n=3). Seminal plasma proteins were analyzed in pools, and differential proteomics was performed using Multidimensional Protein Identification Technology. Progressive motility was lower at 120hours of storage in low resistance, compared to high resistance boars (P<.05). Acrosome and plasma membrane integrity were not affected by the boar category, storage time, or their interaction (P≥.104). Sixty-five proteins were considered for differential proteomics. Among the differentially expressed and exclusive proteins, the identification of proteins such cathepsin B, legumain, and cystatin B suggests significant changes in key enzymes (eg, metalloproteinases) involved in spermatogenesis, sperm integrity, and fertilizing potential. Differences in the seminal plasma suggest that proteins involved in the proteolytic activation of metalloproteinases and proteins related to immune response modulation could disrupt key cellular pathways during spermatogenesis and epididymal maturation, resulting in altered resistance to chilling injury. Further in vivo studies focusing on the immunological crosstalk between epithelial cells and gametes might explain how the immune regulators influence sperm resistance to hipothermic storage.

Assessment of Immunological Response and Impacts on Fertility Following Intrauterine Vaccination Delivered to Swine in an Artificial Insemination Dose.

To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo. Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2–0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Machine learning to further improve the decision which boar ejaculates to process into artificial insemination doses

Current artificial insemination (AI) laboratory practices assess semen quality of each boar ejaculate to decide which ones to process into AI doses. This decision is aided with two, world-wide used, motility parameters that come available through computer assisted semen analysis (CASA). This decision process, however, still results in AI doses with variable and sometimes suboptimal fertility outcomes (e.g., small litter size). The hypothesis was that the decision which ejaculates to process into AI doses can be improved by adding more data from CASA systems, and data from other sources, in combination with a data-driven model. Available data consisted of ejaculates that passed the initial decision, and thus, were processed into AI doses and used to inseminate sows. Data were divided into a training set (6793 records) and a validation set (1191 records) for model development, and an independent test set (1434 records) for performance assessment. Gradient Boosting Machine (GBM) models were developed to predict four fertility phenotypes of interest (gestation length, total number born, number born alive, and number of stillborn piglets). Each fertility phenotype was considered as a numeric and as a binary outcome parameter, totaling to eight different fertility phenotypes. Data used to further improve the decision process originated from four sources: 1) CASA information, 2) boar ejaculate information, 3) breeding value estimations, and 4) weather information. These data were used to create seven prediction sets, where each new set added parameters to the ones included in the previous set. The GBM models predicted fertility phenotypes with low correlations (for numeric phenotypes) and area under the curve values (for binary phenotypes) on the test data. Hence, results demonstrated that a combination of more data and GBM did not enable further improvement of the AI dose quality checks, resulting in the rejection of our hypothesis. However, our study revealed parameters affecting boar ejaculate fertility which were not used in today’s decision process. These parameters (listed in the top 10 in at least four GBM models) included one parameter associated with boar ejaculate information, two with breeding value estimations, five with CASA information, and one with weather information. These parameters, therefore, should be further investigated for their potential value when assessing the quality of boar ejaculates in daily routine AI doses processing.

Stallion Sperm Integrity After Centrifugation to Reduce Seminal Plasma Concentration and Cool Storage for 4 days

The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 106 sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator for the remainder of the cooling period. At day 0, 2, and 4, total motility (TM), progressive motility (PM), and plasma (PLM) and acrosomal membrane integrity were assessed. Centrifuged groups had higher TM and PM at day 4 than the control group (P < .05). Likewise, centrifuged groups had higher intact PLM in day 4 (P < .05). A single centrifugation cycle to reduce seminal plasma concentration will suffice to preserve sperm integrity acceptable for an artificial insemination dose up to 4 days of cool storage.