Cervical Artificial Insemination Research Articles

Lipidomic profiling of cervical mucus reveals the potential role of pro-inflammatory derived metabolites on sperm transport across the ovine cervix

Internationally, cervical artificial insemination (AI) in sheep yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway where vaginal AI of frozen-thawed semen to a natural oestrus yields non-return rates in excess of 60%, which has been attributed to the ewe breed used in Norway. This study used both metabolomics and an RNA-sequencing approach to assess the lipid production and composition from cervical mucus and tissue of four European ewe breeds (n = 28–30 ewes per breed) with previously reported differences in pregnancy rates following cervical AI with frozen-thawed semen. These breeds included Suffolk (exhibiting low fertility), Belclare (medium fertility) as well as Norwegian White Sheep and Fur (both with high fertility and pregnancy rates > 60%) at both a synchronised and natural oestrous cycle. The aim was to explore the differences between ewe breeds in the lipidomic profile and to identify candidate biomarkers associated with an optimal environment for cervical sperm transport. The results revealed the identification of 255 lipids, of which 170, 102 and 83 were different between ewe breeds, types of cycle and affected by their interaction, respectively (P < 0.05). Reduced levels of lipids involved in the resolution of inflammation (i.e. 14-HDoHE,17-HDoHE, 15-HETE) were identified in the low-fertility Suffolk breed compared to high-fertility ewe breeds. However, there was an up-regulation of the COX pathway accompanied by increased levels of prostaglandins in the Suffolk breed. These findings indicated a sub-optimal and pro-inflammatory environment that could have a negative effect on cervical sperm transport.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

Failure of isoxsuprine HCl to increase sheep fertility after cervical versus laparoscopic AI using chilled semen at different durations

To improve the fertility of cervical artificial insemination (AI) in sheep, we investigated isoxsuprine HCl usage on the cervical passage during cervical AI. We also compared cervical and laparoscopic AI fertility results of using chilled semen at different durations. Semen was collected from rams and diluted as 20 × 106 or 400 × 106 spermatozoa/straw for laparoscopic and cervical AI, respectively, and chilled to 4°C within 2 h. Sheep were inseminated with chilled semen for 8 or 24 h via the laparoscopic or cervical AI method. Moreover, some of the cervical inseminated sheep were injected intramuscularly with 0.5 mg/kg of isoxsuprine HCl 15 min before AI. As a result, the use of isoxsuprine HCl did not affect cervical transit and fertility. In addition, fertility was affected by the storage duration of the semen; laparoscopic AI was more successful than cervical AI in terms of fertility; if cervical AI is performed, the duration between semen collection and AI should be less than 8 h after chilling the semen at 4°C, and if laparoscopic AI is performed, the time between semen collection and insemination can be up to 24 h after chilling the semen at 4°C. Longer storage periods should be studied.

Artificial Insemination: A Review

After the issue of practical Human insemination and its application to humans was resolved, remarks and worries surfaced that didn't seem appropriate given the significance or frequency of the process. Endoscopic artificial insemination is a sophisticated reproductive method that was first used on small ruminants, in which semen is injected into the uterus using a specialized device. By using endoscopic artificial insemination techniques in place of natural mating methods and Trans cervical artificial insemination techniques for locally produced progeny, this research seeks to boost the mass population of Indonesian indigenous. The conditions of artificial insemination have completely changed as a result of the development of semen storage, particularly with regard to application flexibility and safety. Even if fresh semen is still used, it is now evident that semen preservation will play a significant role in artificial insemination going forward. Increasing the gamete density at the fertilization location is there asoning behind artificial insemination. Alot of procedures might cause a miss formed pregnancy, which could damage the physical entity of the pregnancy from the moment it formed—in the womb or in a test tube—until it was delivered, so one of a pregnancy's rights is tube preserved and protected until the baby is born, and that conception can happen even in the absence of natural spouse contact using techniques like artificial insemination which will accelerate the rate of genetic improvement. &amp; to conceive a woman using a method other than sexual contact by Artificial Insemination.

Comparative evaluation of natural tupping with fixed-time laparoscopic and cervical insemination techniques using chilled semen in estrous synchronized sheep

The present study was conducted in crossbred ewes to compare the efficiency of different artificial insemination techniques with natural tupping following fixed time artificial insemination using fresh semen. Crossbred ewes (n=29) were randomly divided into three groups with 10 animals in natural tupping (NT) and laparoscopic artificial insemination (LAI) groups and nine animals in cervical artificial insemination (CAI) group. Ewes in all the treatment groups were subjected to the same estrus induction protocol: insertion of intravaginal progesterone sponges for 10 days followed by an intramuscular injection of 500 IU eCG at the time of sponge withdrawal. Immediately after removal of sponges, animals of NT group were kept with proven breeding rams up to 72 h. Fixed time cervical or laparoscopic insemination was done at 48 h after sponge removal. The insemination was repeated 12 h later in CAI group. The pregnancy and lambing rates were significantly higher in NT group (90%, 90%), than CAI group (55.5%, 55.5%) and LAI group (20%, 10%). The prolificacy rate was significantly higher in CAI group (180%) than NT group (111.1%) and LAI group (100%). The serum progesterone concentration was higher in pregnant ewes on day 10, day 17 and day 35 than non-pregnant ewes. The fixed time cervical insemination following intravaginal progesterone sponges for 10 day+eCG protocol resulted in better pregnancy rate and prolificacy rate in crossbred ewes during breeding season.

Metabolic signature of cervical mucus in ewe breeds with divergent cervical sperm transport: a focus on metabolites involved in amino acid metabolism

IntroductionCervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used.Objectives and methodsThis study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility).ResultsA total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset (P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively (P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS (P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds (P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle.ConclusionThe suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Timed artificial insemination in Serrana nanny-goats: the effects of vaginal specula and artificial insemination technicians

The success of artificial insemination (AI) depends on the equipment used, especially regarding the facilitation of artificial insemination technicians’ work and the positioning of insemination guns in the genital tract of animals. This study aimed to evaluate how vaginal specula and artificial insemination technicians affected the fertility rates of timed artificially inseminated Serrana Transmontano goats (which are indigenous to Portugal). For this, 58 adult nanny-goats aged between three and nine years were used. They were reproductively controlled by a short progestogen treatment (FGA) (seven days) with equine chorionic gonadotropin (eCG). Timed cervical AI (43 hours after the end of the hormonal treatment) was performed with chilled semen. Nanny-goats were inseminated by two artificial insemination technicians (A vs. B) alternating two vaginal specula (Minitub vs. “Reyes”). Pregnancy was diagnosed by ultrasound 41 days later. About 98% of nanny-goats responded to our treatment with FGA and eCG. In total, 82.8% of nanny-goats were pregnant 41 days after AI. Neither vaginal specula (Minitub: 81.2% vs. “Reyes”: 84.6%) nor artificial insemination technicians (A: 82.8% vs. B: 82.8%) affected fertility rates. Thus, we conclude that neither the vaginal specula used in this research nor the artificial insemination technicians affected Serrana goat pregnancy rates.

Fixed-time artificial insemination in Churra Galega Bragançana ewes:

This work was carried out with the objective of studying the effect of the anti-reflux device for ovine insemination (DARIO) on the fertility rate in ewes of the native Portuguese breed Churra Galega Bragançana (CGB) artificially inseminated at fixed-time. Eighty-one ewes aged between one and five years were used. At the end of April 2021, ewes’ reproductive control was carried out, using a short progestagen treatment (five days) + equine chorionic gonadotropin (eCG). Cervical artificial insemination (AI) was performed at fixed-time (55 hours after the end of the hormonal treatment), with cooled semen. All ewes were inseminated by the same inseminator: 39 ewes with the DARIO tip on the end of the sheath and 42 without this tip. The pregnancy diagnosis was made by ultrasonography 41 days after AI. Age and body condition (BC) did not affect the ewes’ response to the applied hormonal treatment or the fertility rate. Approximately 95.1% of the ewes responded to the progestagen + eCG treatment. Forty-one days after AI, 86.4% of the ewes were pregnant. The use of the DARIO tip did not affect the fertility rate.

The Role of Housing Conditions on the Success of Artificial Insemination in Intensively Reared Dairy Ewes in Greece.

Simple SummaryArtificial insemination (AI) is a powerful tool for animals’ genetic improvement. However, its application in sheep remains relatively limited due to inconsistent results, since the success of the method is dependent on many factors. In our study, we investigated the impact of housing conditions during the summer period on the fertility of dairy sheep in Greece, after cervical AI with cooled semen. The pregnancy rate was found to be negatively affected by the ambient temperature and Temperature-Humidity Index inside the shed, space availability, frequency of bedding renewal, and outdoor access. Appropriate housing conditions could help to increase pregnancy rates following AI during the summer months.The objective was to assess the effect of housing conditions during the summer months on the success rates of cervical artificial insemination (AI) with cooled semen, in intensively reared dairy ewes in Greece. The study involved 2083 Lacaune ewes from 23 flocks that were serviced during May to September. An estrous synchronization protocol with the insertion of progestogen sponges for 14 days and eCG administration at sponge removal, was used. All ewes were inseminated 54–57 h after sponge removal with cooled semen (15 °C) from 10 Lacaune rams. Pregnancy diagnosis was performed via trans-dermal ultrasonography at 35–40 days after AI. Data recording started the day after sponge placement (15 days prior to AI), and lasted up to 14 days after AI. Daily records included temperature, relative humidity, and Temperature-Humidity Index (THI) inside the shed. Available space and volume per animal, frequency of bedding renewal, access to a yard, and indoor light were also recorded in each farm. Binary logistic regression of data records showed that temperature and THI increases at days −15 to +4 around AI (day 0) had a negative effect on pregnancy rates (reducing the likelihood of pregnancy by 3–6% and 7%, respectively). The latter also decreased significantly (p < 0.05) in farms with high stocking density, non-frequent bedding renewal, and outdoor access by ewes (by 30%, 34%, and 44%, respectively). Overall, the results indicate that appropriate housing conditions are warranted to increase the success of AI in dairy ewes during the summer months.

Effect of L-arginine and Eugenol on Ram Semen Kinematic Parameters and Post Thawed Fertility Rate after Trans-cervical Artificial Insemination

Backgroung: Long term storage of spermatozoa is one of the most desirable tools of assisted reproductive technologies. The purpose of this research was to evaluate the effect of two antioxidants on sperm kinematic parameters after the freezing-thawing process and the post thawed fertility rate after the trans cervical artificial insemination. Methods: Ejaculates from Lacune rams were collected using artificial vagina, were pooled and diluted using based extender (Biladyl, Minitube) and based extender with antioxidants (Eugenol, Arginine). Trans-cervical artificial insemination was performed with frozen seminal material in straws. Result: No significant differences were observed in samples cryopreserved in a base extender and in the base extender supplemented with Arginine. In Eugenol supplemented extender the kinematic parameters were significantly higher for p=0.5 (30.15% progressive motility after thawing) than in the others groups (23.57%, 24.38%). The fertility rate was higher for the straws conserved in extender supplemented with Eugenol (80%) compared with the base extender (55.55%) and the base extender supplemented with Arginine (60%).

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport†.

Worldwide, cervical artificial insemination using frozen–thawed semen yields low pregnancy rates. The only exception to this is in Norway, where vaginal insemination with frozen–thawed semen yields pregnancy rates in excess of 60% and which has been attributed to the specific ewe breed used. Our previous work demonstrated differences in cervical gene expression at the follicular phase of the estrous cycle in ewe breeds with known differences in pregnancy rates. In this study, we characterized the cervical transcriptome of the same ewe breeds [Suffolk, Belclare, Fur, and Norwegian White Sheep (NWS)] during the luteal phase, as an optimal environment at the luteal phase could better prepare the cervix for sperm migration through the cervix at the subsequent follicular phase. High-quality RNA extracted from postmortem cervical tissue was analyzed by RNA sequencing. After stringent filtering, 1051, 1924, and 611 differentially expressed genes (DEGs) were detected in the low-fertility Suffolk breed compared with Belclare, Fur, and NWS, respectively. Gene ontology analysis identified increased humoral adaptive immune response pathways in Suffolk. Increased expression of multiple immune genes supports the presence of an active immune response in the cervix of Suffolk ewes, which differentiates them significantly from the other three ewe breeds. Inflammatory pathways were upregulated in the Suffolk, resulting in higher expression of the potent pro-inflammatory cytokines. Therefore, higher levels of pro-inflammatory cytokines indicate unresolved inflammation in the cervix of the low-fertility Suffolk breed that could contribute to reduced cervical sperm transport in the next follicular phase.

Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle

BackgroundCervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds).ResultsRNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P < 0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds.ConclusionsThis novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Biochemical and molecular characterization of sialylated cervical mucins in sheep

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen–thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Comparison of Five Protocols of Estrous Synchronization on Reproductive Performance of Hu Sheep.

The purpose of this study is to compare five protocols of estrous synchronization for Hu ewes to obtain the most effective and economical protocol, to apply the advantageous scheme in large-scale sheep farming. Healthy multiparous Hu ewes (n = 150) were randomly divided into five groups, and all ewes were administered fluorogestone acetate (FGA, 45 mg) vaginal sponge. The sponges of the first three groups (Groups I, II, and III) were removed on the 11th day, and 0.1 mg of PGF2α was injected intramuscularly on the ninth day. Group I received 6 μg of gonadotropin-releasing hormone (GnRH) by intramuscular injection at 36th h after withdrawal of the sponge. Group II was injected 330 IU of pregnant mare serum gonadotropin (PMSG) on the ninth day. The combination of 6 μg of GnRH and 330 IU of PMSG was treated in Group III at the same time as Group I and Group II. The sponges of the latter two groups (Groups IV and V) were removed on the 13th day, and 330 IU of PMSG was injected intramuscularly simultaneously. PGF2α (0.1 mg) was administered on the 12th day in Group IV. All ewes were detected for estrus at 24, 36, 48, 60, and 72 h after the sponge removal. The loss of sponge and vaginitis was recorded when the sponge was withdrawn. Cervical artificial insemination (AI) was performed with fresh semen of Dorper rams diluted with skimmed milk. After 30 days of insemination, the conception was detected with a veterinary B-ultrasound scanner. The lambing status of all ewes and the cost of drugs for estrous synchronization in each group were recorded. The results showed the following: (1) on the whole, the average percentage of estrous ewes in the period of 24–36 h and 36–48 h after removal was significantly higher than other three periods and that of the period of 60–72 h was significantly lower than the first three periods after removal; (2) there was no significant difference in percentages of estrous ewes in any of the five time periods, sponge loss rate, vaginitis rate, total percentage of estrous ewes, conception rate, single lambing rate, twinning rate, and multiple lambing rate of ewes among five protocols; (3) total percentage of estrous ewes and conception rate were more than or equal to 80% in the Groups II and III, and the twinning lamb rate of the Group II protocol was 70%; (4) there was no difference in lambing rate of ewes among Groups II, III, IV, and V; (5) the Group III had the highest drug cost of 22.5 CNY. In conclusion, considering the lambing rate, twinning lamb rate, and drug cost for estrous synchronization, Group II was the most advisable for application and promotion in large-scale sheep farms among these five protocols of estrus synchronization.

Conserved and breed-specific differences in the cervical transcriptome of sheep with divergent fertility at the follicular phase of a natural oestrus cycle

BackgroundThe outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle (n = 8 to 10 ewes per breed).ResultsHigh-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering (P < 0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds.ConclusionsThese breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine.

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (>52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers—the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa—which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates.

PSV-8 Effects of Air Exposure and Agitation on Quality of Stored Boar Semen Samples

Abstract Recent adoption of post cervical artificial insemination facilitates lowered concentrations of semen inseminated. In order to achieve this reduced concentration, the volume of the insemination dose has been reduced and replaced with additional air in the semen storage container. The objective of this study was to investigate the effects of semen volume, air contact inside semen dose tubes, daily agitation of semen doses and extender type on semen quality, thermo-resistance and bacterial growth in extended boar semen doses over 7 days of liquid storage at 17 C. Ejaculates from 4 terminal cross-bred boars were collected for 4 weeks and used in the 3 x 2 x 2 factorial study. The effects of treatment (CON: 80ml doses sealed at the top of the tube; 40HIGH: 40ml doses sealed at top of tube; and 40LOW: 40ml doses sealed at top of the liquid), extender type (long-term vs short-term), and agitation (agitated vs not agitated) were investigated. The result of the study revealed that motility (P=0.014) and viability (P=0.007) in 40HIGH were lower than CON. pH (P&amp;lt; 0.001) was higher in 40HIGH compared to CON. Agitation did not impact motility (P=0.541), viability (P=0.406) or morphology (P=0.970) while long-term extender maintained higher motility (P=0.034), viability (P&amp;lt; 0.001) and normal acrosomes (P&amp;lt; 0.001) compared to short-term extender. VAP (P=0.039) of 40HIGH was lower than CON in a thermo-resistance test. Bacteria were cultured on both sheep’s blood and MacConkey agar and neither treatment (P=0.798; 0.766) nor agitation (P=0.396; 0.476) impacted bacterial growth in this study. In conclusion, semen doses prepared with 80mL or 40mL of total volume with minimal air contact in the tubes yield more desirable semen quality, as air contact negatively impacts boar semen pH and sperm motility. Additionally, regardless of volume or air exposure, daily agitation of boar semen doses did not affect semen quality.

Reproductive performance in gilts following applications of different insemination doses and techniques

This study evaluated the applicability of intrauterine artificial insemination (IUAI) in gilts and the impact of age at insemination and different body characteristics of gilts on the success rate for cannula insertion. Additionally, reproductive performance was evaluated for IUAI and cervical artificial insemination (CAI), considering different semen dose sizes. A total of 636 gilts were assigned in a 2 × 2 factorial design: two artificial insemination techniques (CAI and IUAI) and two semen dose sizes (1.5 × 109 sperm cells/50 mL or 2.5 × 109 sperm cells/80 mL). In those gilts assigned to IUAI (n = 319) the success rate for intrauterine cannula insertion was evaluated according to weight at first detected estrus, body condition score (BCS), and age at insemination. Reproductive performance, occurrence of bleeding, and semen backflow during all inseminations were compared among groups (2 × 2). Two subgroups were evaluated regarding the time expended to perform insemination (n = 380), and the semen backflow collected during 1 h after insemination (n = 114). The success rate for intrauterine cannula insertion, based on a successful insertion in all inseminations performed during estrus, was 58.9%. Additionally, greater possibility (>60%; P ≤ 0.04) of cannula insertion was observed in heavier gilts (≥124 kg), as well as in older gilts (≥225 d) and those with greater BCS (>3). There were no differences among the groups in pregnancy rate (≥95.3%; P = 0.23), farrowing rate (≥93.7%; P = 0.54), total piglets born (≥14.5; P = 0.45), as well as, bleeding (P = 0.48) and backflow (P = 0.48) during insemination. However, the percentage of semen backflow volume and percentage of sperm cells in the backflow were lower in gilts inseminated by CAI with 1.5 billion sperm cells/50 mL (P < 0.01) than in the other groups. There was no expressive reduction in time expended to perform IUAI compared to CAI. However, gilts inseminated with 1.5 billion sperm cells/50 mL showed a lower total time to inseminate than all other groups (P < 0.01). In conclusion, higher weight, BCS, and age increased the success rate for cannula insertion. However, IUAI did not optimize the insemination procedure, and remains limited for gilts due to the low success rate for cannula insertion. Reproductive performance was not affected by IUAI or CAI using 1.5 or 2.5 billion sperm cells in 50 or 80 mL, respectively, suggesting the possibility of using CAI with 1.5 billion sperm cells/50 mL in gilts.