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Abstract Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development.

Abstract In the current era of genomic selection, there is an increased demand to collect semen from genomically selected sires at a young age. The objective of this study was to assess the effect of bull age, ejaculate number, and season of collection on semen production (ejaculate volume, sperm concentration, and total sperm number; TSN) and sperm motility (prefreeze and post-thaw total and gross motility) parameters in Holstein Friesian bulls in a commercial artificial insemination (AI) center. The study involved the interrogation of a large dataset collected over a 4-yr period, (n = 8,983 ejaculates; n = 176 Holstein Friesian bulls aged between 9 mo and 8 yr). Bulls aged less than 1 yr had the poorest semen production and sperm motility values for all parameters assessed compared with bulls older than 1 yr (P < 0.01). First ejaculates had greater semen production and greater prefreeze motility values than second consecutive ejaculates (P < 0.01), but despite this, there was no difference in post-thaw motility. When subsequent ejaculates were collected from bulls aged less than 1 yr, semen production and sperm motility did not differ compared with mature bulls. Semen collected in winter was poorest in terms of sperm concentration and TSN, but best in terms of post-thaw motility (P < 0.01). In conclusion, second ejaculates can be collected, particularly from bulls aged less than 1 yr, without a significant decrease in post-thaw sperm motility, thus may be a useful strategy to increase semen availability from young genomically selected AI bulls in high demand.

The critically endangered kākāpō ( Strigops habroptilus ) has suffered population declines due to habitat loss, hunting, and predation. Conservation efforts, including translocation to predator-free islands, have helped increase numbers of this flightless parrot from 51 individuals in 1995–142 in 2019. However, low fertility and high embryo mortality, likely due to genetic bottlenecks continue to hinder population growth. This is further aggravated by the kākāpō’s lek mating system, which allows only a minority of males to father a disproportionate number of offspring, resulting in 21% of non-reproductive males. The study aimed to enhance assisted reproduction techniques to assess male fertility, increase egg fertility, and ensure genetic diversity. Artificial insemination (AI) was used to mimic a second copulation, as females mating with multiple males show higher fertility rates. During the 2019 breeding season, semen collection was successful in 20 males and in 93.5% of 46 attempts using abdominal massage method and electric stimulation technique. Semen volume, colour, consistency, contamination, pH and the motility, concentration, viability and morphology of spermatozoa were analysed. Ejaculate volume ranged between 0.1 and 210 µl and the mean pH was 7.5 ± 0.4 ( x ― ± SD). Average sperm viability was 87.4 ± 10.0% with a total motility of 60.9 ± 22.0% and a progressive motility of 28.3 ± 19.8%. AI was performed 15 times in 12 females, improving second clutch fertility (70% vs. 29.4% without AI). Egg fertility in the second clutch without AI was 29.41% (5/17) compared to 70% (14/20) after AI. Paternity testing confirmed AI offspring (four chicks of three females), including from two previously non-reproductive males, enriching the gene pool with rare alleles (e.g., genes from Fiordland founding population). This study demonstrates the value of assisted reproduction in conserving endangered avian species by improving reproductive success and preserving genetic diversity.

Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.

French artificial insemination companies have been running a marker-assisted selection program since 2001 to determine which young bulls should be progeny tested. A first batch of 899 Holstein sires receiving their first proofs based on progeny daughters has been studied. Estimated breeding values with or without marker information were computed based on information available in April 2004, and correlated to daughter yield deviations available in 2007 for production traits. Marker-assisted estimated breeding values presented greater correlations with daughter yield deviations than those calculated using only pedigree index. The average improvement in correlation was 0.043 and ranged from +0.001 for protein yield to +0.103 for fat percentage. This gain was based on the initial and suboptimal conditions of the program and is expected to increase in the coming years because of several improvements implemented since the start of the marker-assisted selection program.

Background The preservation of epididymal spermatozoa is useful for saving important genetic material from valuable individuals who die suddenly or have to be castrated. The birth of puppies after artificial insemination with canine epididymal spermatozoa has been reported in only a few cases. Surgical insemination with frozen-thawed epididymal spermatozoa has resulted in pregnancies, but usually with low conception rates. Freshly collected and chilled epididymal canine semen has also resulted in conception after vaginal insemination. Considering the invasiveness of surgical insemination and the almost unlimited storage time of cryopreserved spermatozoa, transcervical intrauterine insemination with frozen-thawed epididymal spermatozoa would be beneficial. It has the potential to use genetic material that would otherwise be lost, both in domestic dogs and for the preservation of wild threatened canids. Case presentation A 7-year-old, 20 kg male hunting dog was injured by a wild boar during hunting, and euthanasia was recommended for welfare reasons. Because the dog was a hunting champion in a numerically very small breed, the owner wanted to have spermatozoa preserved for future breeding. The dog was anaesthetised, both testes were removed, and the dog was thereafter euthanized. Spermatozoa from both caudae epididymides were released in a prewarmed Uppsala extender with the mincing method. The samples were routinely frozen with the Uppsala method. A half-filled straw was used for test thawing, resulting in 20% motile spermatozoa with slow progressive movement. Three years later, a 23-month-old bitch of the same breed was inseminated with endoscopically guided transcervical intrauterine sperm deposition. She was inseminated once, five days after a serum progesterone value of 6.9 nmol/mL was reached, and two days after a value of 24.8 nmol/mL was reached. The total amount of cryopreserved spermatozoa was used (a total dose of 1087 × 10 6 spermatozoa and 217 × 10 6 progressively motile spermatozoa remaining after test thawing). Eight puppies were born 59 days after insemination. Conclusions Although rarely reported, artificial insemination with cryopreserved epididymal canine spermatozoa is an alternative in preserving valuable genetic animals when a male is injured beyond recovery.

Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08 × 10(-59)). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development. Summary Sentence Bacterial-induced endometritis impairs conceptus development by altering histotroph composition and conceptus gene expression in dairy cattle. Graphical Abstract

This study evaluated the impact of pre-freezing semen dilution rate and dimethyl acetamide (DMA) concentration on the post-thaw motility and fertility of cryopreserved rooster sperm. Rooster ejaculates were diluted with a standard EK extender to achieve low (LSC; 1 × 10⁹ sperm/mL) and high (HSC; 2 × 10⁹ sperm/mL) sperm concentrations. Each dilution group was further treated with three DMA concentrations (3%, 6%, or 9%) before cryopreservation. Post-thaw sperm motility traits were obtained by computer-assisted sperm analysis (CASA), and fertility features were evaluated through artificial insemination in hens. The current results showed that HSC significantly improved total motility (TM), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF), but reduced linearity (LIN) and straightness (STR) compared to LSC. DMA concentration had a quadratic effect on motility, with 6% yielding the highest progressive motility (PM), straight line velocity (VSL), and BCF. Fertility outcomes revealed that HSC resulted in higher fertilization rates, while neither DMA concentrations nor their interaction with dilution rates exerted significant effects on fertility traits. VCL, ALH, and BCF showed positive correlations with pipping-chicks rates, whereas STR, LIN, and WOB displayed negative correlations. These findings underscore the critical interplay between dilution rate and cryoprotectant concentration and provide practical guidance for developing more reliable cryopreservation protocols that can be applied under field conditions to enhance fertility management in poultry production.

Stringent efforts are being made to restrict the development of antimicrobial resistance by good antibiotic stewardship in medical, veterinary and environmental health. In the pig industry, considerable volumes of antibiotic-containing extender are utilised in artificial insemination. Separating spermatozoa from bacteria by colloid centrifugation followed by cold storage could provide an alternative to antibiotics in semen extenders, provided that sperm fertility is not affected by this combination of techniques. The objective of this proof-of-concept study was to compare reproductive outcomes in sows inseminated with control sperm samples or with samples prepared by Single Layer Centrifugation (SLC) through a low density colloid and cooled to 4 °C. A further aim was to compare the SLC sperm preparations with and without antibiotics. Sows were divided into three groups for insemination as follows: control semen prepared and stored conventionally ( n  = 16), SLC samples prepared with antibiotics and cold stored ( n  = 18), SLC samples prepared without antibiotics and cold stored ( n  = 18). The yield of spermatozoa was 84–93%, respectively. Farrowing rates were 88% for controls and 89% for SLC samples. Mean litter size was 16.6, 17.5 and 17 for controls, SLC with antibiotics and SLC without antibiotics, respectively. Stillborn piglets per litter were also similar between groups (0.7, 1.4 and 1.4 respectively). In conclusion, the combination of techniques did not adversely affect sperm fertility or reproductive outcomes of inseminated sows, regardless of whether antibiotics were included in the resuspended sperm pellets after SLC.