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The aims of this study were I) to compare the follicular diameter, corpus luteum diameter and serum progesterone (P4) concentrations in cows treated with conventional protocol vs. injectable P4 protocol; II) to determine the serum P4 profile in ovariectomized heifers; and III) to compare pregnancy rate between protocols. In experiment I, multiparous cows received a protocol for ovulation synchronization with an intravaginal P4 device (n = 38; device + EB day 0; device removal + PGF2α + eCG + EC day 8) or injectable P4 (n = 38; injection + EB day 0; PGF2α + eCG + EC day 8). In experiment II, ovariectomized heifers (n = 8) were treated with injectable P4 and blood samples were collected to determine the serum P4 profile. In experiment III, multiparous cows were timed AI with two different P4 approaches, intravaginal P4 device (n = 48) or injectable P4 (n = 47). In the first experiment, cows treated with P4 device had higher (P < 0.05) diameter of dominant follicle after ovulation induction (11.6 ± 1.8 vs.10.3 ± 1.8 mm) and ovulation rate (97%, 37/38 vs. 47.3%, 18/38) than cows treated with injectable P4. But, the follicular growth daily was higher (P < 0.05) in cows treated with injectable P4 than intravaginal device (1.3 ± 0.4 vs. 1.0 ± 0.3 mm/day, respectively). In experiment II, the P4 concentration peak occurred within 48 hours (6.54 ng/mL) and decreased after 96 hours (P < 0.05) after P4 injection. In experiment III, cows with P4 device had higher (P < 0.05) pregnancy rate than the injectable P4 group (60.4 vs. 34.0%, respectively). These results demonstrate that although the intravaginal P4 devices showed a higher pregnancy rate, a protocol with injectable P4 represents an easier method and a promising alternative for TAI in cattle.

Non-lethal methods for semen collection from elasmobranchs to better understand species reproduction has accompanied the development of artificial insemination. Ejaculates (n = 82) collected from whitespotted bamboo sharks Chiloscyllium plagiosum (n = 19) were assessed and cold-stored raw or extended at 4 °C. Females (n = 20) were inseminated with fresh or 24-48 h cold-stored raw or extended semen and paternity of offspring determined with microsatellite markers. Insemination of females with fresh semen (n = 10) resulted in 80 hatchlings and 27.6% fertility. Insemination of females with semen cold-stored 24 h (n = 4) and 48 h (n = 1) semen resulted in 17 hatchlings and fertilization rates of 28.1% and 7.1% respectively. Two females inseminated with fresh or cold-stored semen laid eggs that hatched from fertilization and parthenogenesis within the same clutch. Parthenogenesis rate for inseminated females was 0.71%. Results demonstrate artificial insemination with cold-stored semen can provide a strategy for transport of male genetics nationally and internationally, precluding the need to transport sharks. Production of parthenotes in the same clutch as sexually fertilized eggs highlights the prevalence of parthenogenesis in whitespotted bamboo sharks and poses important considerations for population management.

High levels of reproductive failure were detected in some Spanish sow farms in the Spring of 2010. Regular returns to estrus and variable reductions in litter size were observed. The problem started suddenly and did not appear to be related to the quality of the ejaculates, disease, alterations of body condition or any other apparent reasons. Subsequent studies determined that the problem was the origin of the plastic bags used for semen storage. Chemical analysis of the suspicious bags identified unexpected compounds such as BADGE, a cyclic lactone and an unknown phthalate that leached into the semen at concentrations of 0.2 to 2.5 mg/L. Spermatozoa preserved in these bags passed all of the routine quality control tests, and no differences were observed between storage in the control and suspicious bags (p > 0.05). In vitro fecundation tests and endocrine profiler panel analysis (EPP) did not show any alterations, whereas the in vivo tests confirmed the described failure. This is the first described relationship between reproductive failure and toxic compounds released from plastic bags.

The rapid spread of the African swine fever virus (ASFV), causing severe disease with often high fatality rates in Eurasian suids, prevails as a threat for pig populations and dependent industries worldwide. Although advancing scientific progress continually enhances our understanding of ASFV pathogenesis, alternative transmission routes for ASFV have yet to be assessed. Here, we demonstrate that ASFV can efficiently be transferred from infected boars to naïve recipient gilts through artificial insemination (AI). In modern pig production, semen from boar studs often supplies many sow herds. Thus, the infection of a boar stud presents the risk of rapidly and widely distributing ASFV within or between countries. Daily blood and semen collection from four boars after intramuscular inoculation with ASFV strain 'Estonia 2014' resulted in the detection of ASFV genomes in the semen as early as 2 dpi, in blood at 1 dpi while semen quality remained largely unaffected. Ultimately, after insemination with extended semen, 7 of 14 gilts were ASFV positive by 7 days post insemination, and all gilts were ASFV positive by 35 days post insemination. Twelve out of 13 pregnant gilts aborted or resorbed at the onset of fever. A proportion of fetuses originating from the remaining gilt showed both abnormalities and replication of ASFV in fetal tissues. Thus, we present evidence for the efficient transmission of ASFV to gilts via AI and also to implanted embryos. These results underline the critical role that boar semen could play in ASFV transmission.

We evaluated the reproductive performance of sows after single fixed-time AI under a tropical climate and investigated the influences of season and insemination technique on the efficacy of single fixed-time AI. After weaning, the sows were divided into CONTROL (n = 212) and FIXED-TIME (n = 212) groups. Sows in the CONTROL group were inseminated at 12 and 36 h after the onset of oestrus, while sows in the FIXED-TIME group were administered 10 μg of GnRH at 72 h after weaning and were inseminated 32 h later. Reproductive performance parameters, including total born, born alive, mummified foetuses and stillborn piglets per litter, piglet birth weight, variation of piglet birth weight within litter, regular return-to-oestrus and farrowing rate, were compared between the two groups. Season was classified into two groups: cool (n = 170) and hot (n = 254), and insemination technique was classified into two groups: conventional AI (n = 171) and intra-uterine insemination (IUI) with a reduced number of spermatozoa (n = 253). On average, regular return-to-oestrus (3.3 vs. 5.6%, P > 0.05) and farrowing rates (92.8 vs. 88.1%, P > 0.05) did not differ between CONTROL and FIXED-TIME groups. However, the total born and born alive piglets per litter in the FIXED-TIME were lower than in the CONTROL group (12.0 vs. 12.8 piglets/litter; P = 0.030 and 11.3 vs. 12.2 piglets/litter, P = 0.007). Interestingly, the number of total born piglets in the FIXED-TIME group was lower than in the CONTROL group only in the sows inseminated in the hot season (11.7 ± 0.32 and 12.9 ± 0.31, respectively, P = 0.005). Piglet birth weight, variation of piglet birth weight within litter, number of piglets at weaning and body weight of piglets at weaning did not differ between groups, irrespective of the season (P > 0.05). The total number of piglets born per litter in the FIXED-TIME group was lower than that in the CONTROL group in sows inseminated via IUI (11.7 ± 0.32 and 12.9 ± 0.32, respectively, P = 0.013), but not in sows inseminated using conventional AI (12.7 ± 0.42 and 12.5 ± 0.41, respectively, P = 0.772). Single fixed-time AI could be successfully performed in sows under a tropical climate, with a promising reproductive performance. However, a decreased litter size at birth after single fixed-time AI was observed when insemination was performed in the hot season. Moreover, single fixed-time AI using IUI with a reduced number of spermatozoa also decreased litter size at birth.

We developed a simple, noninvasive artificial insemination technique to study epigenetic germline inheritance in mice. This technique avoids interfering factors introduced by superovulation, surgery, in vitro culture or mating that can confound the transmission of acquired epigenetic information through the germline. Using a stress model, we demonstrate that our method is suited to test the causal involvement of the male germline in transmitting acquired information from father to offspring.

Mycoplasma agalactiae (Ma) is the main causative agent of ovine contagious agalactia, which is a serious disease of small ruminants. In endemic areas, its most common clinical situation consists of chronically infected herds, and asymptomatic infected individuals represent an epidemiological risk regarding the transmission of this disease. The aim of this work was to detect the presence of asymptomatic rams infected with Ma in different artificial insemination centers, and to determine the most effective way to identify these individuals so as to implement adequate surveillance protocols. For this purpose, 215 rams and 14 teaser sheep were sampled taking auricular, nasal, and vaginal swabs and serum samples. In addition, ejaculates from 147 rams were analyzed. These samples were subjected to specific culture and molecular techniques to isolate and identify mycoplasmas, and to a serological test to detect antibodies against Ma. Mycoplasma agalactiae was detected in 47 (4.4%) of the 1077 samples analyzed, and also one individual resulted seropositive. Thus, 37 (17.2%) of the 215 studied rams were infected with Ma. The specimens which proportionally yielded the greatest number of positive results for this pathogen were semen samples (13.6%), followed by nasal swabs (5.8%). In contrast, the sampling of the external auricular canal and the serological analyses resulted insufficient to effectively detect infected individuals. Asymptomatic rams infected with Ma were detected in all the analyzed artificial insemination centers, highlighting the need to implement adequate surveillance protocols to prevent the presence of these individuals in these centers, reducing the risk of transmitting contagious agalactia.

The paper described a novel technique for semen collection in large psittacines (patent pending), a procedure which was not routinely possible before. For the first time, a large set of semen samples is now available for analysis as well as for artificial insemination. Semen samples of more than 100 psittacine taxa were collected and analysed; data demonstrate large differences in the spermatological parameters between families, indicating an ecological relationship with breeding behaviour (polygamous versus monogamous birds). Using semen samples for artificial insemination resulted in the production of offspring in various families, such as Macaws and Cockatoos, for the first time ever. The present technique represents a breakthrough in species conservation programs and will enable future research into the ecology and environmental factors influencing endangered species.

In this study, we aimed to determine risk factors associated with cytological endometritis (CYTO) diagnosed at artificial insemination (AI) in dairy cows. The CYTO risk factors were evaluated based on 1.625 AI-CYTO samples obtained from 873 Holstein-Friesian cows from in total 18 dairy herds in Flanders (Belgium). The endometrial cytology samples were obtained using the cytotape technique, which consisted of adapting a 1.5 cm piece of paper tape to a standardly loaded AI catheter, covered with a double guard sheet. The polymorphonuclear cells' (PMNs) cut-off point for CYTO at AI was set at ≥ 1%. We constructed multilevel generalized mixed effect models in order to identify the risk factors associated with the presence of CYTO at AI. The CYTO prevalence at AI was 27.8% at the animal level, while the within-herd level prevalence ranged from 10.7 to 39.7%, with an average of 28.1%. Risk factors associated with the occurrence of CYTO were parity ≥2 [odds ratio (OR) = 1.8], days in milk (DIM) at AI ≥ 124 (OR = 0.4), and warm months of the year [July (OR = 2.9), August (OR = 2.3), and September (OR = 1.4)]. In conclusion, the present study supports that multiparous cows and cows that are inseminated in the summer months have a higher risk to suffer from CYTO at insemination, while the risk for CYTO is lower when the insemination is taking place at ≥ 124 DIM.

This study was conducted to evaluate various factors affecting fertility following insemination of dromedary camels. In experiment 1, camels were either bred by natural mating (NM) or inseminated in the body of uterus with whole, split (50:50) or 1 mL of undiluted ejaculate. In experiment 2, camels were inseminated with fresh diluted semen either in the body of the uterus or tip of the uterine horn and at either the time of ovulation induction (0 h), 24 or 30 h later. In experiment 3, camels were inseminated at the tip of the uterine horn with different doses of fresh diluted semen (75, 150 or 300 x 106 motile spermatozoa) or with 150 x 106 motile spermatozoa diluted with different extenders (Green buffer, Optixcell or Triladyl). In experiment 4, camels were inseminated in the tip of the uterine horn with diluted (Triladyl or Optixcell) liquid-stored semen or diluted (Triladyl) frozen-thawed semen consisting of either 300 or 500 x 106 motile spermatozoa. The pregnancy rate in camels bred by NM was similar to camels inseminated with whole undiluted ejaculates whereas insemination with 1 mL undiluted ejaculate resulted in lower pregnancy compared to whole and split undiluted ejaculates (P < 0.05). Deposition of semen in the uterine body resulted in lower pregnancy rates compared to deposition in the tip of the horn (35.3% versus 72.2%, P < 0.05) but insemination at the time of ovulation induction and 24 h later resulted in higher pregnancy rate to camels inseminated at 30 h after induction (68.4 and 70.0% versus 23.5%; P < 0.05). Artificial insemination with 75 x 106 motile spermatozoa resulted in lower pregnancy rates compared to 150 and 300 x 106 motile spermatozoa doses (40.9% versus 65.2 and 70.0%, respectively) and pregnancy rate was not affected by extenders. Insemination of chilled motile spermatozoa stored in either Triladyl or Optixcell resulted in similar pregnancy rates, regardless of insemination dose, although an upward trend with increasing sperm number was apparent (Triladyl; 11.1% versus 21.1% and Optixcell; 5.9% versus 12.5%, for 300 x 106 and 500 x 106 groups, respectively; P > 0.05). No pregnancies were obtained with frozen thawed semen. In conclusion, this study demonstrated that the success of camel AI is highly dependent on sperm dose, location of semen deposition, timing of insemination and semen type. Further studies are required to determine the reason for the compromised fertility of preserved semen despite apparent high in vitro quality.

This cohort study investigates the association between COVID-19 vaccination status and artificial insemination by partner outcomes among couples experiencing infertility in China.

The profitability of the dairy and beef industries is largely affected by the actually achieved reproductive efficiency. Although a large proportion of cows worldwide are bred by artificial insemination (AI) services, many potential factors affecting the outcome of pregnancy by AI remain to be addressed. In the present study, we investigated the vaginal microbiota by high-throughput sequencing of 16S rRNA gene and analyzed their association with differential pregnancy outcomes (i.e., pregnant vs. nonpregnant) of multiple AI services in dairy cows. Sequencing of the V3-V4 region totally produced 512,046 high-quality sequences that were computationally clustered into 2,584 operational taxonomic units (OTUs). All OTUs were taxonomically assigned to 10 bacterial phyla. There were statistically significant differences among the three AI service times (T1, T2 and T3) with respect to the Shannon index and number of observed OTUs (p < 0.05). Bray-Curtis distance-based PCoA analysis also revealed that T2 group could be significantly distinguished from T1 and T3. However, no significant difference between the pregnant and nonpregnant cows was found in confidence regarding both alpha diversity and beta diversity. These results could help us better understand the possible influence of vaginal microbial community on pregnancy outcomes of AI service in cows.

There has been development of an antiretrograde flow device (DARIO), for sheep cervical artificial insemination (CAI). There, however, needs to be optimization of sperm volume and concentration of insemination doses when the DARIO is used for CAI. Objectives were to compare fertility rates (proportion of ewes lambing as a result of CAI) when there was use of the DARIO for CAI: two sperm volumes containing equal numbers of spermatozoa: 0.25 mL of 1,600 × 106 spermatozoa/mL and 0.50 mL of 800 × 106 spermatozoa/mL (Test 1 group), and two sperm volumes with a different number of spermatozoa/AI dose: 0.25 mL and 0.50 mL of 1,600 × 106 spermatozoa/mL (Test 2 group). There were 335 ewes from seven farms assigned to 60 batches (equally divided into a Control and Test 1 group). For the Test 2 group, 462 ewes from nine farms were assigned to 88 batches (equally proportioned into Control group and Test 2 groups). For the Test 1 group, proportion of ewes lambing as a result of CAI were 0.701 ± 0.2679 and 0.595 ± 0.2393 for the Control and Test 1 groups, respectively (P = 0.163). For the Test 2 group, proportions of ewes lambing were 0.550 ± 0.2598 and 0.658 ± 0.2412 for the Control and Test 2 group, respectively (P = 0.041). An inclusion of a larger number of spermatozoa per insemination in a 0.50 mL dose volume resulted improved proportion of ewes lambing as a result of CAI when there was used of the DARIO.

The African catfish is one of the most promising warm-freshwater species for aquaculture development in the upcoming years, although there are two primary limitations in its production: less-than-desirable survival rates and low larvae quality. In this study, a novel method of egg fertilization for this species was evaluated which has already been successfully utilized in the production of other finfish. The results indicate that dividing the semen designated for fertilization of eggs into smaller aliquots and adding it to the eggs at 30 s intervals (i.e., 0, 30 and 60 s), after activation of the eggs with water, increased the hatching rate percentage (97.1%) compared to control groups (87.9%) in which the entire portion of semen was added to eggs at the time of water addition for egg activation. Results with repeated evaluations in the field confirmed that eggs with greater biological quality were fertilized at a similar rate using the modified and control methods for fertilization, although when the eggs were of a lesser biological quality, the fertilization rate was greater using the modified methods than the control methods.

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group's high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Fixed-time post-cervical artificial insemination (FTPCAI) allows a wider use of high indexing boars and a considerable reduction in labour requirements in swine production. The aim of this study was to evaluate fixed-time artificial insemination (FTAI) efficiency using different artificial insemination protocols and porcine luteinising hormone (pLH) to induce ovulation. A total of 597 weaned sows in which oestrus detection was performed once daily (08:00 am) was allocated to three groups: FTPCAI1 (n=199) - sows received a 5-mg (4 ml) intramuscular injection of pLH at oestrus onset, and were inseminated 24h later; FTPCAI2 (n=199) - sows received 5mg of pLH and were inseminated at oestrus onset (0 h) and 24h after; and MultPCAI (n=199) - sows did not receive pLH, and the first AI was performed at oestrus onset (0 h) and repeated every 24h during oestrus. Homospermic doses (1.5 × 10(9) total sperm cells/50 ml) were used in post-cervical artificial insemination (PCAI) in all the treatments. Hormonal treatment did not affect (P>0.05) the interval between oestrus onset and ovulation (overall 32.4h) and there were no differences (P>0.05) in farrowing rate (overall 91.6%) or litter size (overall 12.6 piglets born) among treatments. In sows treated with pLH at oestrus onset, a single PCAI with 1.5 billion sperm cells did not compromise reproductive performance compared with a double PCAI at 24h intervals.

Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI).

Introduction: Sperm storage within the uterovaginal junction (UVJ) of avian species occurs in specialized structures termed sperm storage tubules (SSTs) and allows for prolonged storage of semen, though the molecular mechanisms involved in semen preservation are not well understood. Little work has been done examining how function of the SSTs is impacted by insemination and by semen present in the SSTs. Methods: Transcriptome analysis was performed on isolated SSTs from turkey hens receiving no insemination (control), sham-insemination, or semen-insemination at three timepoints (D1, D30, and D90 post-insemination). Bioinformatic and functional annotation analyses were performed using CLC Genomics Workbench, Database for Annotation, Visualization, and Integrated Discovery (DAVID), and Ingenuity Pathway Analysis (IPA). Pairwise comparisons and k-medoids cluster analysis were utilized to decipher differential expression profiles in the treatment groups. Results: The SST transcriptome of the semen inseminated group exhibited the greatest differences within the group, with differences detectable for up to 90 days post insemination, while control and sham-inseminated groups were more similar. In the semen-inseminated samples, upregulation of pathways relating to classical and non-classical reproductive signaling, cytoskeletal remodeling, physiological parameters of the local UVJ environment, and cellular metabolism was observed. In the sham-inseminated samples, upregulation of immune pathways and non-reproductive endocrine hormones was observed. Discussion: This work provides insights into the molecular level changes of the SST in response to insemination as well as to the presence of semen. Results from this study may have direct implications on fertility rates as well as potential strategies for avian semen cryopreservation protocols.

Oxidative stress occurs when there is greater than optimal production of reactive oxygen species (ROS) or an antioxidant system failure. Calves produced using in vitro fertilization (IVF) or cloning (CA) have greater mortality rates, with greater incidence of respiratory diseases, which could be explained by the deleterious outcomes from oxidative stress. Calves were studied that were produced using: artificial insemination (AI; n = 20), in vitro fertilization (IVF; n = 15) or cloning (CA; n = 15). Blood samples were collected at 6, 12, 24 and 48 h subsequent to the time of birth. The cloned calves had greater ROS production from lipid peroxidation, with greater thiobarbituric acid reactive substances. This factor was associated with a lesser amount of superoxide dismutase in the CA. Calves produced using IVF had a greater activity of catalase and glutathione peroxidase, either due to greater production of hydrogen peroxide or greater efficiency of enzymatic response of these neonates. Calves produced using AI had greater concentrations ​​of reduced thiol groups. These associated factors may indicate there is greater oxidative stress in calves produced by IVF and cloning than with use of AI, however in these calves there was an effective response to these oxidative stressors within 48 h subsequent to birth. Hence, calves produced using IVF and by cloning have greater ROS production when compared to calves produced using AI. The calves produced using IVF, however, had a greater enzymatic activity or were more efficient in adapting to ROS when compared to calves produced by cloning.