Artificial Insemination Research Articles

Evidence-based practice in canine artificial insemination.

A number of methods are currently used to predict the optimal date of insemination of the breeding bitch, particularly with the use of frozen-thawed canine semen which has a far shorter lifespan than fresh semen. Aside from confirming cytological oestrus, vaginal cytology is of no assistance in predicting the most fertile day(s) in a bitch; however, a neglected avenue of research suggests that vaginal cytology may be of great importance in confirming the days of optimal fertility retrospectively. Similarly, vaginoscopy provides clues as to the stage of a bitch's cycle but is inadequate as a sole determinant of her most fertile days. Nevertheless, vaginoscopy is useful to identify very late oestrus and the onset of dioestrus, as well as Stage I of labour (cervical dilatation). Due to variations in the rate at which circulating progesterone concentrations rise in individual bitches, the reliability of circulating progesterone concentrations for determining the optimal day(s) of insemination with frozen-thawed semen decreases as values rise. Moreover, progesterone assay results can vary widely due to extrinsic factors such as the time of blood sampling, sample storage conditions and the assay employed. Finally, this review investigates evidence surrounding various insemination routes and suggests that well-performed vaginal insemination, even with frozen-thawed semen, may be an acceptable approach for cases where transcervical insemination is impractical.

Use of long-acting injectable progesterone in fixed-time artificial insemination protocols

Knowledge of the physiology of the estrous cycle in bovine females has been extremely important in monitoring and improving strategies in the use of drugs to control their estrous cycle and follicular development, providing better recruitment, selection, growth, and maturation until ovulation, with strategies to further optimize estrus and ovulation synchronization protocols. The objective of this work is to carry out a bibliographical analysis comparing the use of slow-release progesterone devices in relation to the use of long-acting injectable progesterone, taking into account their advantages, disadvantages and characteristics within a fixed-time artificial insemination program.

The veterinarian’s role in reducing GHG emissions from beef and dairy production

The beef and dairy industries make significant contributions to sustainability objectives by providing our society with nutri­tious food, supporting farmers’ livelihoods, generating billions of dollars in economic activity, and contributing to healthy ecosystems. However, cattle production also generates negative environmental impacts, including greenhouse gas (GHG) emis­sions. Veterinarians have a unique opportunity to interface with beef and dairy farmers to assist with the implementation of products and practices that reduce GHG emissions, such that their clients can be more profitable and more sustainable. In general, the GHG intensity of beef and dairy production can be reduced by increasing output yields of beef and milk, while re­ducing inputs and maximizing efficiency. Some of the specific interventions include: early entry of feeder cattle to a feedlot, dry manure management, optimized inorganic nitrogen fertil­izer use, increased fat in the ration, use of anti-methane feed additives and ionophores, sourcing low-carbon corn, select­ing genetics for high feed efficiency, raising moderate/small mature beef cows and feed efficient dairy cows, improving pregnancy rate and reducing dystocia, conducting pregnancy checks, implementing artificial insemination, promoting herd health products and practices to reduce death loss, using growth-promoting implants, and utilizing SOP Lagoon®. As a trusted source of scientific information, veterinarians can be a conduit of information for producers and contribute to the ongoing effort to improve the resiliency of the beef and dairy industries.

Side of chute does not affect the sensitivity of Tritrichomonas foetus sample collection in bulls when using dominant hand for collection

Tritrichomonas foetus, a sexually transmitted disease in cattle, continues to plague the cattle industry despite years of control programs. One reason for this continued prevalence through­out the U.S. and the world is that accurate diagnosis is compli­cated in the fact that retrieval of organisms for diagnosis and subsequent testing may be compromised by multiple factors in­cluding, but not limited to, how samples are collected. A commonly used sampling technique for diagnosing T. foetus in bulls is scraping of the preputial and penile mucosa with ei­ther an artificial insemination pipette or specially designed T. foetus testing device such as the Pizzle Stick Trich Testing De­vice (Lane Manufacturing Denver, CO). Parker et al. noted that right-hand dominant practitioners were more commonly suc­cessful in retrieving T. foetus in known positive bulls when col­lecting from the right side of the bull compared to the left; how­ever, the opposite was not investigated for left-hand dominant practitioners and only cultures were used to declare whether a sample was positive or not. The objective of this study was to determine if using current RT-qPCR testing if there is a differ­ence in rate of positive tests pending side of chute and use of dominant hand for testing.

Short Communication: Higher abundance of 2-dehydro-d-gluconate in the plasma of sub-fertile or infertile Bos taurus heifers.

Infertility or subfertility impacts approximately 5% and 15% of dairy and beef heifers (Bos taurus), respectively. Heifers that do not produce a calf within an optimum window of time have a significant negative impact on the profitability and sustainability of the cattle industry. Selection of heifers based on their fertility potential remains a challenge yet to be resolved. Here, we tested the hypothesis that heifers of different fertility potential have differing metabolome signatures in their plasma. We obtained blood from Bos taurus heifers at their first artificial insemination and processed the samples to separate the plasma. The heifers were classified based on their reproductive outcome as fertile (pregnant and delivered a calf after their first artificial insemination (AI)) or sub-fertile (Angus heifers: no pregnancy after two AI and exposure to a bull; Holstein heifers: no pregnancy by the third AI). We tested the relative abundance of 140 metabolites obtained from 22 heifers (Angus fertile n = 5, Angus sub-fertile n = 7, Holstein fertile N = 5, Holstein sub-fertile N = 5). The metabolite 2-Dehydro-D-gluconate (C6H10O7) was significantly more abundant in the plasma of sub-fertile heifers in both breeds (1.4-fold, false discovery rate <0.1). In the context that a small proportion of circulating metabolites in the plasma were quantified in this study, the results show that the metabolomic profile in the blood stream may be associated with heifer fertility potential.

Segmentation of mature human oocytes provides interpretable and improved blastocyst outcome predictions by a machine learning model

Within the medical field of human assisted reproductive technology, a method for interpretable, non-invasive, and objective oocyte evaluation is lacking. To address this clinical gap, a workflow utilizing machine learning techniques has been developed involving automatic multi-class segmentation of two-dimensional images, morphometric analysis, and prediction of developmental outcomes of mature denuded oocytes based on feature extraction and clinical variables. Two separate models have been developed for this purpose—a model to perform multiclass segmentation, and a classifier model to classify oocytes as likely or unlikely to develop into a blastocyst (Day 5–7 embryo). The segmentation model is highly accurate at segmenting the oocyte, ensuring high-quality segmented images (masks) are utilized as inputs for the classifier model (mask model). The mask model displayed an area under the curve (AUC) of 0.63, a sensitivity of 0.51, and a specificity of 0.66 on the test set. The AUC underwent a reduction to 0.57 when features extracted from the ooplasm were removed, suggesting the ooplasm holds the information most pertinent to oocyte developmental competence. The mask model was further compared to a deep learning model, which also utilized the segmented images as inputs. The performance of both models combined in an ensemble model was evaluated, showing an improvement (AUC 0.67) compared to either model alone. The results of this study indicate that direct assessments of the oocyte are warranted, providing the first objective insights into key features for developmental competence, a step above the current standard of care—solely utilizing oocyte age as a proxy for quality.

Designing a diagnostic method to predict the optimal artificial insemination timing in cows using artificial intelligence

Dairy farmers and beef cattle breeders aim for one calf per year to optimize breeding efficiency, relying on artificial insemination of both dairy and beef cows. Accurate estrus detection and timely insemination are vital for improving conception rates. However, recent challenges such as operational expansion, increased livestock numbers, and heightened milk production have complicated these processes. We developed an artificial intelligence (AI)-based pregnancy probability diagnostic tool to predict the optimal timing for artificial insemination. This tool analyzes external uterine opening image data through AI analysis, enabling high conception rates when inexperienced individuals conduct the procedure. In the initial experimental phase, images depicting the external uterine opening during artificial insemination were acquired for AI training. Static images were extracted from videos to create a pregnancy probability diagnostic model (PPDM). In the subsequent phase, an augmented set of images was introduced to enhance the precision of the PPDM. Additionally, a web application was developed for real-time assessment of optimal insemination timing, and its effectiveness in practical field settings was evaluated. The results indicated that when PPDM predicted a pregnancy probability of 70% or higher, it demonstrated a high level of reliability with accuracy, precision, and recall rates of 76.2%, 76.2%, and 100%, respectively, and an F-score of 0.86. This underscored the applicability and reliability of AI-based tools in predicting optimal insemination timing, potentially offering substantial benefits to breeding operations.

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

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

131 DNA methylation profile in bovine fetal liver is affected by maternal vitamin and mineral supplementation during early gestation

Abstract Early gestation is the critical period for successful pregnancy establishment. During this period, maternal nutrition affects fetal development with potentially long-lasting consequences on offspring performance. Despite the known effects of vitamins and minerals on embryonic development, supplementation is still not a widely adopted practice. Here we focused on the effects of maternal vitamin and mineral supplementation from pre-breeding to d 83 of gestation. We hypothesized that the DNA methylation pattern of genes involved with fetal hepatic metabolism and function would be altered in response to vitamin and mineral supplementation during early gestation. Sixteen Angus crossbred heifers (~16 mo of age) were randomly assigned to either a treatment (vitamin and mineral supplementation, VTM, n = 8) or control (corn-based carrier without supplementation, NoVTM, n = 8) group. Supplemented heifers received 0.45 kg •heifer-1•d-1 of to provide 113 g of vitamin-mineral premix (Purina Wind &amp; Rain Storm All-Season 7.5 Complete, Land O’Lakes, Inc., Arden Hills, MN) from d 71 to 148 before breeding to d 83 of gestation. All heifers received 100% of NRC recommended nutritional requirements targeting 0.28 kg •heifer-1•d-1 of gain. The heifers were bred through artificial insemination following a 7-d Co-Synch + CIDR estrus synchronization protocol. All pregnant heifers were surgically ovariohysterectomized on d 83, and liver samples were collected from the fetuses. DNA methylation profiles from 16 fetuses (8 per treatment) were determined using Reduced Representation Bisulfite Sequencing (RRBS). The analysis was conducted using methylKit pipeline where data was normalized using principal components. We identified 794 differentially methylated cytosines (DMCs) for the control (NoVTM) vs VTM comparison. Most of the DMCs were found in introns followed by intergenic, promoter, and exonic regions. Among these DMCs, 60% (479) were hypermethylated and 40% (315) were hypomethylated, potentially regulating 739 nearby genes (FDR &amp;lt; 0.1). Biological processes over-represented by these genes included transport of ions through voltage gated channels (KCNK13, KCNT1, CACNA1B, KCNK9, LRRC55, KCNAB2, CACNA1S, KCNJ2, CACNG5, CLIC5, TPCN2, KCNQ1), which may be associated to pregnancy establishment by inducing pregnancy-associated relaxation and uterine artery dilatation. Some genes associated with structural development were also regulated by VTM supplementation (i.e., TIAM1, COL18A1, DYSF, BMP10, RXRA, EHMT1, COL4A2, PLEC, ZFAT, MTMR2, NAV1, ANHX, ACACB, and LIMK2). Collectively, these results suggest that periconceptual maternal vitamin and mineral supplementation affects the methylation pattern and potentially regulates the expression of genes involved in fetal liver development and ion transport.

Voltage-dependent anion channels are involved in the maintenance of pig sperm quality during liquid preservation

Pigs are usually bred through artificial insemination with liquid semen preserved at 15–20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 μM and 50 μM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 μM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 μM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.

283 Identification of new or existing risk factors associated with the development of sow pelvic organ prolapse

Abstract A survey was conducted between March and December of 2022 to examine the variation between farms that had high and low pelvic organ prolapse (POP) rate. The objective was to identify new or existing risk factors associated with the development of POP, which could open new avenues for further investigation. Sow farms (n = 53) across 26 production systems located in the US, Canada, and Mexico were used. Sampling of farms was not random. High and low POP systems were selected within each region as well as high and low POP farms within each system. Over 800 variables related to general farm information and management, performance traits, reproductive management, replacement gilt management, gestation and farrowing practices, labor and health management, and nutrition were collected during farm visits. To be included in the analysis, at least 70% of observations were needed for continuous variables with at least 10% of observation for each category for categorical variables. Univariable models were fitted to identify variables associated with annualized POP rate for 2021. Variables with P ≤ 0.10 were selected for further analysis, with 94 predictor variables selected for principal components analysis (PCA). A factor analysis of mixed data was conducted, and results were then fitted into a hierarchical clustering on PCA, which segregated the data into three clusters: 2 with low POP (2.5%) and 1 with high POP (6.1%) prevalence (low vs high P &amp;lt; 0.05). The resulting clusters explained 33.7% of the variability in annualized 2021 POP rate. Differences between clusters were evaluated by Kruskall-Wallis test for numerical variables and chi-square test for categorical variables. High POP cluster farms had greater total piglets born over the years, greater number of sows per worker, greater annual gestation feed usage, and a lower mean body condition score at 16 wk of gestation compared with low POP cluster farms. High POP cluster farms had a decreased proportion of farms using conventional artificial insemination, a greater proportion of farms utilizing cup waterers in gestation for sows in group housing and in farrowing, and greater proportion of farms with a routine sleeving practice. High POP cluster farms compared with low POP cluster farms had greater gestation distillers dried grains with solubles (DDGS) inclusion level (14.6% vs 4.0%, SE 3.02), greater levels of standardized total tract digestibility (STTD) P% (0.49% vs 0.39%, SE 0.014), narrower analyzed Ca to STTD P ratio (1.7 vs 2.2, SE 0.06), and lower phytase inclusion and release (0.16% vs 0.09%, SE 0.014). These variables were also the predictors with the greatest R2 values in the univariable models. These observations suggest further research is needed to evaluate the connection between dietary Ca and P and pelvic organ prolapse.

242 Comparison of GnRH administration day in the 7&amp;7 Synch versus 8&amp;6 Synch Protocol for fixed-timed artificial insemination in beef heifers

Abstract The research aimed to investigate the impact of reducing the time interval for progesterone exposure on the ovulatory follicle just before performing fixed-timed artificial insemination (FTAI). This was achieved by adjusting the timing of administering a gonadotropin-releasing hormone agonist (GnRH; specifically, 100 µg gonadorelin, known as Cystorelin). The study assessed the effectiveness of two estrous synchronization protocols, the “7 &amp; 7 Synch” and a modified protocol, by examining estrus expression (EE) and the resulting pregnancy rates (PR) following FTAI. The research utilized frozen conventional Angus semen from Select Sires, Inc. (Plain City, OH), and involved 66 Angus-cross heifers from the beef cattle research unit near Fayetteville at the University of Arkansas. To ensure consistency, the heifers were divided into two treatment groups based on their cyclicity status (CYC) and age. CYC status was determined by assessing a reproductive tract score (RTS) 1 wk before the synchronization process. RTS scores ranged from 1 to 5, with RTS 1-3 indicating pre- or peripubertal status and scores of 4 and 5 indicating cycling. Heifers with an RTS of 1 and 2 were excluded from the study before synchronization commenced. Both treatment groups involved the insertion of a controlled internal drug-releasing device (CIDR; specifically, Eazi-breed, containing 1.38 g of progesterone) into the vagina on d -14. Simultaneously, a prostaglandin F2α analogue (PGF; 500 µg cloprostenol sodium, known as Synchsure) was administered. In the first treatment group (7&amp;7), GnRH was administered on d -7, while in the second group (8&amp;6), GnRH was given on d -6. In both cases, PGF was administered on d 0 when the CIDR was removed, precisely 72 ± 1 h before FTAI, along with another dose of GnRH. Estrus expression (EE) was assessed during FTAI by examining Estrotect patches (Rockway Inc., Spring Valley, WI). EE was considered present when a patch had 50% or more of its color rubbed off, and it was deemed absent when less than 50% of the color had worn off. Pregnancy rates (PR) were determined through ultrasound examination on d 55. EE and PR were analyzed by treatment using the GLIMMIX procedure on SAS 9.4 (SAS Institute Inc., Cary, NC). Statistical differences were declared at α ≤ 0.05 and tendencies declared between 0.05 &amp;lt; P ≤ 0.1. Heifers on the 8&amp;6-d protocol had greater rectal temperatures at breeding compared with 7&amp;7-d heifers (P &amp;lt; 0.0001). AI pregnancy rates were greater in heifers on the 8&amp;6-d protocol compared with the heifers on 7&amp;7-d protocol (P = 0.625) with average pregnancy rates of 39.4% and 30.3%, respectively. Overall, pregnancy rate was greater as well for the 8&amp;6-d heifers compared with the 7&amp;7-d heifers (P = 0.044), with mean overall pregnancy rates of 78.8% and 54.5%, respectively.

243 Effects of body weight change in pre-production Duroc boars on semen collection training, semen production parameters, and longevity in a commercial boar stud

Abstract Our objective was to assess the effects of percentage body weight (BW) change in developing Duroc boars on semen collection training, semen production parameters, and longevity in a commercial boar stud. Retrospectively, 164 boars were divided into one of three groups based on their percentage BW change from arrival to the boar stud until the end of the 42-d isolation period. The one-third of boars that had the greatest percentage of BW change during the isolation period gained 36.1% to 10.1% (TOP). The middle one-third of boars that were intermediate in percentage BW change during isolation gained 9.7% to 2.6% (MIDDLE). The final group consisted of one-third of boars that either minimally gained or lost BW (2.5% to -9.5% change in BW; BOTTOM). During the isolation period boars in the TOP group lost the least amount of backfat (indicated by ultrasound or caliper score; P &amp;lt; 0.05), while boars in the BOTTOM group lost the most backfat. Average boar age at the time of successfully becoming a working boar (successfully mounting the collection dummy and producing an ejaculate with a motility ≥ 70% and percent normal morphology ≥ 65%) and the proportion of boars that were successful at become working boars did not differ between groups (P &amp;gt; 0.05). Among working boars, the concentration of sperm in ejaculates tended to differ (P = 0.0740) between groups (Table). Boars in the TOP group had a greater percentage of sperm with normal morphology (P = 0.0337) than boars in the BOTTOM group. There was a group by production week interaction (P &amp;lt; 0.0001) for semen ejaculate volume, total number of sperm, and total number of sperm with normal morphology. Boars in the TOP and MIDDLE groups produced more total sperm and normal sperm than the BOTTOM group from wk 8 to 20 of being working boars, resulting in production of considerably more doses for artificial insemination. During their time as working boars, removal from the stud by cull or death was not different between groups (P &amp;gt;0.05). There was also no difference (P &amp;gt; 0.05) between groups on the average week of removal from the stud, with each group having boars most frequently removed at wk 22 of production. While percentage BW change during isolation did not affect semen collection training and longevity in the stud, the increase in production of total normal sperm in the TOP and MIDDLE groups compared with the BOTTOM group could have substantial economic benefits for boar stud. Our study suggests that managing boars individually to ensure they stay in a positive metabolic state is ideal for optimal semen production at a boar stud.

238 Effect of electronically controlled floor cooling pad on semen performance of Duroc boars during the summer heat stress

Abstract The objective of the study was to evaluate the effects of using electronically controlled floor cooling pad (ECP) on boar semen production during the summer season in a boar stud in Scottsville, Kentucky. Sixty boars (526.26 ± 188.69 d old) were placed in two rows of pens (2.06 x 0.66 m) in a 350 head tunnel ventilated boar barn and were randomly assigned to either CONTROL (n = 30; no ECP) or PAD (n = 30) treatments. The ECP had 4.25 L cold water flushed through a group of 5 pads connected to each other, when temperature sensors reached 27°C or every 4 mins for sensors placed on 5th pad and 24°C or every 4 mins for sensors placed on 1st pad. Boars experienced natural summer heat stress (daily temperature, humidity and dewpoint: Mean = 24.03°C, 87.9%, 21.7°C, hourly mean high = 28.9°C, 83.4%, 25.8°C) for a period of 30 d from July 15, 2023 to August 14, 2023 wherein the cooling pad remained on and were flushing based on the set points mentioned above. Semen was collected from each boar at least once each week and semen samples were diluted in a commercial semen extender and analyzed for motility using a CASA system, volume using a gram scale, and concentration using a spermiodensimeter. A semen sample was also stained using Hancock stain and evaluated at 100x oil immersion to assess sperm morphology (100 sperm cells analyzed as normal or having retained cytoplasmic droplets, distal midpiece reflex or other head/tail abnormalities). Total sperm production, percentage of morphologically normal sperm, percentage of motile sperm were calculated and used to determine whether the ejaculate met threshold for use in an artificial insemination program or whether the ejaculate was trashed. Data were evaluated using PROC MIXED for repeated measures in SAS (v.9.4) with the main effects of treatment and week, using the age of the boar, and the number of days between collections as covariates, and boar nested within treatment as a random effect. There were no differences between CONTROL and PAD boars for semen production measures (P &amp;gt; 0.05). PAD boars had greater morphologically normal sperm compared with CONTROL boars using Hancock stain (P = 0.0362); however, this effect was lost when the percentage of total normal sperm for the entire ejaculate was calculated (P = 0.56). Overall, maintaining boars on floor cooling pads during the summer months had minimal impacts on semen production and reduced the presence of morphologically abnormal sperm.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Medically Assisted Reproduction and Risk of Cancer Among Offspring

Cancer is a leading cause of death among children worldwide. Treatments used for medically assisted reproduction (MAR) are suspected risk factors because of their potential for epigenetic disturbance and associated congenital malformations. To assess the risk of cancer, overall and by cancer type, among children born after MAR compared with children conceived naturally. For this cohort study, the French National Mother-Child Register (EPI-MERES) was searched for all live births that occurred in France between January 1, 2010, and December 31, 2021 (and followed up until June 30, 2022). The EPI-MERES was built from comprehensive data of the French National Health Data System. Data analysis was performed from December 1, 2021, to June 30, 2023. Use of assisted reproduction technologies (ART), such as fresh embryo transfer (ET) or frozen ET (FET), and artificial insemination (AI). The risk of cancer was compared, overall and by cancer type, among children born after fresh ET, FET, or AI and children conceived naturally, using Cox proportional hazards regression models adjusted for maternal and child characteristics at birth. This study included 8 526 306 children with a mean (SD) age of 6.4 (3.4) years; 51.2% were boys, 96.4% were singletons, 12.1% were small for gestational age at birth, and 3.1% had a congenital malformation. There were 260 236 children (3.1%) born after MAR, including 133 965 (1.6%) after fresh ET, 66 165 (0.8%) after FET, and 60 106 (0.7%) after AI. A total of 9256 case patients with cancer were identified over a median follow-up of 6.7 (IQR, 3.7-9.6) years; 165, 57, and 70 were born after fresh ET, FET, and AI, respectively. The overall risk of cancer did not differ between children conceived naturally and those born after fresh ET (hazard ratio [HR], 1.12 [95% CI, 0.96 to 1.31]), FET (HR, 1.02 [95% CI, 0.78 to 1.32]), or AI (HR, 1.09 [95% CI, 0.86 to 1.38]). However, the risk of acute lymphoblastic leukemia was higher among children born after FET (20 case patients; HR 1.61 [95% CI, 1.04 to 2.50]; risk difference [RD], 23.2 [95% CI, 1.5 to 57.0] per million person-years) compared with children conceived naturally. Moreover, among children born between 2010 and 2015, the risk of leukemia was higher among children born after fresh ET (45 case patients; HR, 1.42 [95% CI, 1.06 to 1.92]; adjusted RD, 19.7 [95% CI, 2.8 to 43.2] per million person-years). The findings of this cohort study suggest that children born after FET or fresh ET had an increased risk of leukemia compared with children conceived naturally. This risk, although resulting in a limited number of cases, needs to be monitored in view of the continuous increase in the use of ART.

Abstract PO4-11-10: Comparison of frozen embryo transfer cycle (FET) outcomes between young women with breast cancer (YWBC) with and without pathogenic variants (PV), and breast cancer-free PV carriers

Abstract BACKGROUND: In vitro fertilization (IVF) and preimplantation genetic testing for aneuploidy (PGT-A) and monogenic disorders (PGT-M) are valuable tools for YWBC with or without PV such as BRCA1/2, ATM, CHEK2, PMS2 to prevent variant transmission to offspring.  However, to date, there is scarce data on the usability of these cryopreserved, tested embryos and on the outcomes of FET cycles within the cancer population. It has been suggested that PV may influence endometrial thickness which is associated with embryo implantation and ultimately FET success rates. Thus, this study aimed to compare reproductive outcomes of FET cycles between YWBC with an identified PV, breast cancer (BC) patients without a PV, and cancer-free PV-carriers who returned to use their cryopreserved oocytes or embryos derived from IVF ± PGT-A/M cycles. METHODS: Patients that underwent either fertility preservation (FP) prior to BC therapy or preventative IVF/PGT-A/M cycles from 2009-2023 were included. All IVF &amp; PGT-A/M was performed at academic IVF centre. Genetic testing was conducted using high resolution next generation sequencing on the Illumina platform. We compared reproductive outcomes between 3 groups: 1) YWBC with a known PV (BCPV), 2) YWBC without a PV (BCNV), 3) Cancer-free patients who had a PV (CFPV). Primary outcomes measured were endometrial thickness, and FET success rates (i.e., ongoing pregnancy or live birth) analyzed by chi-square, t-tests and ANOVA. RESULTS: 185 patients underwent 202 cycles for either oocyte or embryo freezing. Mean age was 33.8±4.6 years (range 21-45) and mean BMI was 24.6±5.0 kg/m2 (range 15.7-42.7). 64 patients had a PV including BRCA1 (n=22), BRCA2 (n=20) and others (n=22): ATM, BARD1, BRIP1, CHEK2, MLH1, P53, PALB2, PDL2, PMS2, PTCH1, &amp; RAD51D. When comparing the BCPV, BCNV &amp; CFPV groups, no significant differences were found in the mean age (33.7 ± 4.9 vs. 36.8 ± 4.3 vs. 34.3 ± 4.4 years, p=0.52), mean BMI (26.2 ± 4.9 vs. 24.3 ± 4.8 vs. 24.3 ± 4.8 kg/m2, p=0.13), and the proportion of smokers (23.1% vs. 35.0% vs. 36.4%, p=0.72). Only 60% of BCNV performed PGT-A due to their lack of PV. As of June 2023, 44 patients (12 in BCPV, 20 in BCNV, and 12 in CFPV) returned to undergo 81 FET cycles (range 1-5/patient). 12/81(14.8%) FETs involved a surrogate. When comparing the BCPV, BCNV &amp; CFPV who had FET, a significant difference was found in the mean age at oocyte retrieval between BCPV &amp; BCNV (32.9±4.4 vs. 37.0±4.0 years, p=0.001) but not with CFPV (33.5±4.4 years). The endometrial thickness at the time of FET between the BCPV, BCNV, and CFPV groups was similar (10.2±1.6 vs. 10.6±2.6 vs. 9.3±1.9 mm, p=0.12), as was the proportion of smokers (25.0% vs. 36.8% vs. 33.3%, p=0.79). YWBC had a mean delay from egg retrieval to their first FET of 37 months. Estrogen receptor positive (ER+) YWBC had a longer delay than ER- patients (40.1±23.8 months vs. 29.6±11.6 months, p=0.25). 29/44 (65.9%) had at least one successful FET and another 5 YWBC conceived naturally or with intrauterine insemination (IUI); the cumulative pregnancy rate was 77.3% (34/44). The clinical pregnancy rate of 81 FETs was 44.4%. The BCPV group (73.7%) had a significantly higher successful outcome than the BCNV (35.3%) and CFPV (35.7%) groups (p=0.01). CONCLUSIONS: FP done at the time of a BC diagnosis is an effective tool to preserve fertility potential. 77% of patients had at least one baby from either IVF, IUI or conceived naturally. We did not find a difference in endometrial thickness in PV carriers. While all YWBC had a delay to their first FET from the time of their retrieval, those who were ER+ had a longer delay than ER- because of their need for endocrine therapy. The BCPV group had the highest FET success rate which is likely due to their younger age and use of PGT-A/M thereby eliminating FETs of abnormal embryos that would fail to implant. The BCNV were older with more untested embryos. The CFPV group transferred euploid embryos and thus the lower success rate is puzzling and needs further investigation. Citation Format: Maya Sharon-Weiner, Amanda Kauffman, Adina Tanen, Samantha Yee, Svetlana Madjunkova, Karen Glass. Comparison of frozen embryo transfer cycle (FET) outcomes between young women with breast cancer (YWBC) with and without pathogenic variants (PV), and breast cancer-free PV carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-11-10.

Abstract GS02-11: Fertility preservation and assisted reproductive technologies (ART) in breast cancer (BC) patients (pts) interrupting endocrine therapy (ET) to attempt pregnancy

Abstract Background: The POSITIVE trial recently demonstrated that temporary interruption of ET to attempt pregnancy does not increase the short-term risk of recurrence in pts with hormone receptor-positive (HR+) BC. Yet, uncertainty exists regarding the safety of ovarian stimulation for embryo/oocyte cryopreservation to preserve fertility in these pts. In addition, very limited data exist regarding safety and efficiency of ART to achieve pregnancy after ET interruption. Methods: POSITIVE is an international, single-arm, prospective trial that enrolled pts ≤ 42 years with stage I-III HR+ BC who had already received 18-30 months of ET prior to enrollment. The use of ART was a predefined 2ry endpoint. We evaluated factors associated with time to pregnancy in a Fine and Gray competing risks model, which included chemotherapy (chemo) ± GnRH analogue (GnRHa), age, prior ET, prior birth, and menstrual status at enrollment. The cumulative incidence of pregnancy at 12 months from enrollment by age was estimated controlling for competing risks. We performed a logistic regression model to evaluate whether ART use was associated with achieving pregnancy, adjusting for age, receipt of chemo ± GnRHa. The cumulative incidence of BCFI events was estimated according to whether or not ovarian stimulation for cryopreservation was performed at diagnosis and prior to enrollment. BC-free interval (BCFI) was defined as the time from enrollment until the first BC event (invasive local, regional, or distant recurrence or contralateral disease). A 24-month landmark analysis was performed to investigate the safety of ART use after enrollment. Median follow-up was 41 months. Results: 497 out of 516 included pts (96%) were followed for at least 6 months and were evaluable for pregnancy status. 368 out of 497 evaluable pts (74%) reported at least one pregnancy. In a multivariable model, young age was the only factor associated with shorter time to pregnancy. The cumulative incidence of pregnancy at 12 months was 64%, 54% and 38% for pts aged &amp;lt; 35, 35-39 and 40-42 years, respectively. 179 pts (36.0%) had undergone ovarian stimulation for embryo/oocyte cryopreservation at diagnosis and prior to enrollment, of whom 68 (37.9%) reported cryopreserved embryo transfer after enrollment. 215 pts (43.3%) reported using any ART after enrollment, of whom 80 (37.2%) underwent ovarian stimulation for in-vitro fertilization (FIVET) or intracytoplasmic sperm injection (ICSI). Other ART used included clomiphene (n=19), intrauterine insemination (n=37) and egg donation (n=17). Pregnancy rates were 82.4% and 67.5% for women who underwent cryopreserved embryo transfer and FIVET/ICSI, respectively. In a multivariate model, cryopreserved embryo transfer was the only ART independently associated with a higher chance of pregnancy (OR:2.41, 95% CI: 1.75-4.95). The cumulative incidence of BCFI events at 3 years was similar for women who underwent ovarian stimulation for cryopreservation at diagnosis and before enrollment, 9.7% (95% CI: 6.0-15.4%), and those who did not, 8.7% (95% CI: 6.0-12.5%). A 24-month landmark analysis showed no impact of ovarian stimulation for FIVET/ICSI after enrollment on BCFI. Conclusions: This is the largest prospective study to investigate fertility preservation and ART in pts with HR+ BC. Young age was the main factor associated with shorter time to pregnancy. Embryo/oocyte cryopreservation at BC diagnosis followed by embryo transfer after ET interruption was the only approach associated with an increased chance of pregnancy. Ovarian stimulation either at BC diagnosis for embryo/oocyte cryopreservation or after enrollment to POSITIVE for FIVET/ICSI did not appear to impact prognosis in the short term. These data are of paramount importance for oncofertility counselling of young BC pts. Citation Format: Hatem Azim, Samuel Niman, Ann Partridge, Isabelle Demeestere, Monica Ruggeri, Marco Colleoni, Cristina Saura, Chikako Shimizu, Anna Saetersdal, Judith Kroep, Audrey Mailliez, Ellen Warner, Virginia Borges, Frédéric Amant, Andrea Gombos, Akemi Kataoka, Christine Rousset-Jablonski, Simona Borstnar, Junko Takei, Jeong Eon Lee, Janice Walshe, Manuel Ruíz - Borrego, Halle Moore, Christobel Saunders, Vesna Bjelic-Radisic, Snezana Susnjar, Fatima Cardoso, Natalie Klar, Tanya Spanic, Kathryn Ruddy, Martine Piccart, Larissa Korde, Aron Goldhirsch, Richard Gelber, Olivia Pagani, Fedro Alessandro Peccatori. Fertility preservation and assisted reproductive technologies (ART) in breast cancer (BC) patients (pts) interrupting endocrine therapy (ET) to attempt pregnancy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS02-11.

Oxytocin, prostaglandin F2α, and scopolamine for uterine involution of dairy cows

The aim of the study was to compare the effect of three substances with ecbolic activity, Oxytocin, Prostaglandin F2α (PGF2α) and Scopolamine, on the uterine involution process in dairy cows and on the resumption of ovarian activity. Eighty bovine were randomly divided in four groups: GROUP C: 20 cows treated, within 24 h of calving, with 5 mL/head of saline solution; GROUP PG: 20 cows treated, within 24 h of calving, with 150 μg/head of d-cloprostenol; GROUP OX: 20 cows treated, within 24 h of calving, with 50 IU/head of oxytocin acetate; GROUP S: 20 cows treated, within 24 h of calving, with 40 mg/q Scopolamine Butylbromide. Each cow was subjected to blood samples to evaluate the Hydroxyproline (HYP) levels, at T0, within 24 h after calving, and T7, T14, T28, 7, 14, and 28 days after calving, respectively. At T14 and T28, an ultrasound examination was performed to measure the diameter of ex-pregnant horn. In all cows, the reproductive indices (days to first service and number of artificial insemination for conception) were evaluated. In all groups, the HYP concentrations have been rising from T0 to T28, with the maximum levels obtained at T28 in the groups PG and S. As regard the diameter of uterine horn, the comparison among the groups showed significant differences only at T28, with lower values in the group PG and S. In group S and PG, the days to first service were less than other groups. Treatment with Scopolamine and PGF2α resulted in better outcomes, evidenced clinically by more efficient uterine involution and faster ovarian recovery.

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well