Artificial Insemination Research Articles

Effect of Different Female Factors on Pregnancy Rates in Artificial Insemination With Donor Sperm Cycles: A Prospective Observational Study

Tubal abnormalities were evaluated on hysterosalpingo-contrast sonography (HyCoSy) using sodium alginate solution as a contrast agent in 18 repeat breeder cattle. Clear contrast enhancement was observed from the uterine horn to the tubal infundibulum in the patent cases, and the percentage of cases with unilateral or bilateral passage disorder was 64.7%. The post-examination artificial insemination conception rate when ovulatory follicles were present on the patent side was 64.3%, which was significantly higher than 12.5% seen when ovulatory follicles were present on the side affected by passage disorder (P < 0.05). The polymorphonuclear leukocyte percentage in the recovered contrast agent was significantly higher on the passage disorder side than on the patent side, whereas there was no significant difference in C-reactive protein concentrations between the patent and passage disorder sides. The present method can be used for diagnosing tubal abnormalities, and chronic inflammatory changes may be related to tubal passage disorder.

The validation and application of an ovine fertility model using standardised in vitro thresholds to predict the likelihood of pregnancy.

Astragaloside IV improves the quality of in vitro postovulatory aged oocytes by decreasing oxidative stress in mice.

Cervical explants alter gene expression and cytokine secretion in response to seminal plasma compared with spermatozoa alone. Results validate the model to study mechanisms behind sperm-immune tolerance in the ovine cervix ex vivo. The ovine cervix presents a unique barrier to frozen-thawed ram spermatozoa, reducing pregnancy following cervical artificial insemination (AI). This is hypothesised to be related to a modified sperm molecular profile following seminal plasma (SP) exposure and cryopreservation, altering communication with the female environment. However, the impact of different sperm types on the cervix itself has never been explored, in part due to the difficulty of profiling these in vivo interactions. Here, an ex vivo cell culture model was used to explore changes in the transcriptome of cervical explants following exposure to spermatozoa and SP. Explants were collected from Merino ewes (n = 6) post-mortem and cultured with epididymal, epididymal + SP, frozen and frozen + SP ram spermatozoa, SP only, or a media control for 6 h. Gene expression was profiled by RNA sequencing. Multiplex ELISAs quantified the abundance of cytokines in culture supernatant. Cytokeratin and H&E staining confirmed the presence of cervical epithelial cells in cultured explants. The presence of epididymal sperm caused the differential expression of 18 genes compared with the media-only control, while the addition of SP to epididymal sperm altered 781 genes. Differentially expressed genes were involved in pathways related to inflammation, signalling, and ATP synthesis. SP exposure to frozen sperm led to significantly increased IL1A and reduced TGFB1 production compared with other treatments. Results show an altered cervical immune response in the presence of ram SP and sperm. Further research is required to determine molecular candidates which facilitate immune tolerance and sperm transit through the cervix.

This review focuses on the biochemical mechanisms of oxidative damage to sperm, the role of antioxidants, as well as the clinical consequences of supplementation for the improvement of sperm fertility. There is growing interest in dog breeding and in methods of maintaining semen quality for natural mating or artificial insemination (AI). Canine sperm are sensitive to oxidative damage. Semen contains endogenous, enzymatic, renewable, and non-enzymatic, and non-renewable antioxidants. However, the excess of reactive oxygen species (ROS) or depletion in antioxidative defense may lead to oxidative stress, causing damage to sperm cells and a decrease in fertility. The possible way to maintain sperm cell fertility potential is supplementation of diet and/or semen extenders with antioxidants. It seems that oral antioxidant supplementation improves the oxidative status and quality of semen and may have a positive effect on the fertility of male dogs with reproductive problems. Many studies point to the potential role of antioxidant supplementation in extenders in protecting canine sperm from oxidative damage during processing. However, only a few studies have assessed the fertilization capacity of supplemented sperm after AI. Intensive studies, including the examination of pro- and antioxidative properties of dog semen as well as the role of antioxidant supplementation to dogs and semen extenders, should be performed, as it is a serious market and breeders need. The results should be related not only to semen analysis but pregnancy rate as the best marker of fertility. Nevertheless, the use of antioxidants in the supportive treatment of fertility disorders in male dogs to improve semen quality and their addition to dog semen extenders to preserve sperm fertility appears to be reasonable.

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

This study aimed to evaluate the effect of platelet-rich plasma (PRP) on sperm motility in men with asthenozoospermia and assess its potential use in intrauterine insemination (IUI). A prospective randomized controlled pilot study was conducted with 40 men diagnosed with asthenozoospermia. Semen samples were divided into two equal parts: one treated with autologous, non-activated PRP and the other serving as control. Sperm motility was assessed at 1, 3, 6, and 24h using a Makler chamber. A two-step centrifugation method was used for PRP preparation, and all samples underwent identical swim-up protocols. Data were analyzed using two-way repeated measures ANOVA. PRP-treated samples showed significantly higher motility than controls at all time points (p < 0.001). PRP also slowed the decline in motility over time. At 24h, motility was 50.5% in the PRP group versus 21.1% in controls. Statistical analysis confirmed normal data distribution. PRP significantly enhances and preserves sperm motility during in vitro incubation. These findings suggest that PRP may serve as a supportive option in sperm preparation for IUI, potentially reducing the need for more invasive techniques. Further studies are warranted to evaluate clinical outcomes.

Semen cryopreservation is an essential technique in artificial insemination (AI) and assisted reproductive technology (ART). The addition of antioxidants to the freezing medium is a promising strategy to reduce cryo-induced damage to sperm cells. While sperm freezing has been extensively studied, this research aimed to improve the post-thaw durability and functionality of sperm. A total of 110 semen samples 55 from fertile and 55 from infertile individuals were collected and processed. After centrifugation, each sample was mixed with a cryoprotectant medium at a 1:1 (v/v) ratio. Three different cryoprotectant formulations (S1, S2, and S3), each containing varying concentrations of protective agents, were evaluated. The samples were then frozen in liquid nitrogen at -196°C. Post-thaw analyses included assessments of sperm count, motility, vitality, morphology, DNA fragmentation, and reactive oxygen species (ROS) levels representing a major strength of the study due to the comprehensive evaluation of sperm quality. The results showed significant differences in DNA fragmentation between fresh and cryopreserved samples. Among the tested formulations, S3 supplemented with citric acid anhydrous and taurine produced the best outcomes. It significantly improved sperm motility and vitality, while effectively reducing oxidative stress and cryodamage. Statistical analysis using one-way ANOVA and Tukey's HSD test revealed significant differences in sperm morphology between fresh and post-thaw S3-treated samples (p < 0.05), with a 95% confidence level. A limitation of the study was the long interval between the use of fresh control samples and the analysis of cryopreserved samples, which may influence comparative accuracy. Despite this limitation, the findings suggest that the S3 formulation is highly effective in preserving sperm quality. These results support its potential integration into fertility preservation protocols in ART. Furthermore, the outcomes may guide clinical practice and support the development of national policies aimed at improving access to advanced cryopreservation techniques as part of comprehensive reproductive healthcare services.

Changes in maternal nutrition in the periconceptional period can have long term effects on the offspring. The aim of the current study was to determine the influence of feeding 15 g of rumen-protected methionine (RP-Met) during the periconceptional period on female progeny during the post-weaning phase until puberty. A total of 114 beef cows were fed a roughage-based diet and randomized to receive corn gluten feed supplemented with 15 g of RP-Met (Smartamine M, Adisseo) or not (CON) from day -7 to + 7 relative to artificial insemination with female sex-sorted semen. Female calves (n = 32) remained with their dams until weaning. A month after weaning, calves were weighed every 14 d, and withers height (WH), body length (BL), and heart girth (HG) were measured during the post-weaning phase. At 420 ± 13 d of age, a 56-d performance test was conducted, and initial body weight (IBW), final body weight (FBW), dry matter intake (DMI), average daily gain (ADG), and residual feed intake (RFI) were measured. At the end of the performance test, the 12th rib back fat thickness (BF), ribeye area (REA), intramuscular fat (IMF), and rump fat thickness (RF) were determined via ultrasound. Data from the post-weaning phase, performance test, and body composition were analyzed as a RCBD using generalized linear mixed models through the MIXED procedure of SAS, with body measurements analyzed as repeated measures. A treatment-by-day interaction (P = 0.003) was observed for HG, with larger HG for RPM from day 308 onwards. Final BW was greater for RPM heifers (CON = 436.13 ± 1.43 kg; RPM = 445.63 ± 1.52 kg; P < 0.01). Also, RPM heifers had a greater ADG than CON heifers (CON = 1.40 ± 0.04 kg; RPM = 1.65 ± 0.04 kg; P < 0.01). No treatment difference (P > 0.05) was observed for DMI (CON = 12.06 ± 0.19 kg/d; RPM = 12.14 ± 0.18 kg/d) or RFI (CON = -0.12 ± 0.15; RPM = 0.11 ± 0.14). There was also no effect of treatment (P > 0.05) on REA or IMF. RF was less for RPM (CON = 1.16 ± 0.07 cm; RPM = 0.91 ± 0.06 cm), and there was a tendency (P = 0.07) for lesser BF in RPM. In conclusion, supplementing dams with RP-Met enhanced FBW of their heifer progeny during the performance test; furthermore, a treatment-by-day interaction tendency was observed for HG. RPM heifers also had reduced RF and a tendency for reduced BF.

Our bone health as an adult is defined by patterns of development in early life, with perturbed growth during fetal and neonatal periods predisposing individuals to poor bone health in adulthood. Studies have identified poor maternal diet during pregnancy as a critical factor in shaping offspring bone development, with significant impacts on adult bone structure and health. However, the association between a father's diet and the bone health of his offspring remains poorly defined. To address this knowledge gap, we fed male C57BL/6 mice either a control normal protein diet (NPD; 18% protein) or an isocaloric low protein diet (LPD; 9% protein) for a minimum of 8 weeks. Using these males, we generated offspring through artificial insemination, in combination with vasectomised male mating. Using this approach, we derived offspring from either NPD or LPD sperm but in the presence of NPD or LPD seminal plasma. Using micro-computed tomography and synchrotron X-ray diffraction, we observed significant changes in offspring femur morphology and hydroxyapatite crystallographic parameters from just 3 weeks of age in offspring derived from LPD sperm or seminal plasma. We also observed that differential femur morphology and hydroxyapatite crystallographic parameters were maintained into adulthood and into a second generation. Analysis of paternal sperm identified a down regulation of 26 osteogenic genes associated with extracellular matrix levels and maintenance, transcription and growth factors and bone ossification. These observations indicate that poor paternal diet at the time of conception affects offspring bone development and morphology in an age and generation specific manner.

This study aimed to investigate the effect of maternal taurine (Tau) supplementation during gestation on the development of brown adipose tissue (BAT) and the thermogenesis of offspring lambs. Sixty ewes were randomly divided into three groups and daily supplemented with 0 g (control group), 2 g (low Tau), and 4 g (high Tau) during the gestation period after artificial insemination. The body surface temperature of newborn lambs was measured daily. At 15 days of age, lambs were slaughtered for sample collection. Compared to the control group, lambs in the high-Tau group exhibited higher body temperatures (p < 0.05). Maternal Tau-treated lambs had higher expression of UCP1 (p < 0.05), a greater number of multilocular brown adipocytes, and increased mitochondrial count in the BAT (p < 0.05). Additionally, there were higher copies of mitochondrial DNA and expression of mitochondrial proteins in the maternal Tau-treated lambs (p < 0.05). Enhanced AMPK and ULK1 activation (p < 0.05) were also observed in the BAT of 15-day-old lambs. Further analysis of the effects of Tau on BAT stromal vascular fraction (SVF) cells revealed that Tau treatment upregulated UCP1 during the brown adipogenic differentiation stages (p < 0.05). Consistently, Tau treatment upregulated mitochondrial biogenesis genes and increased the number of mitochondria (p < 0.05). Tau treatment significantly upregulated PGC1a and SIRT3, as well as activated AMPK and ULK1 (p < 0.05). Mitophagy is initiated, but the mitochondrial morphology remains stable. In conclusion, maternal Tau supplementation during gestation promotes brown adipogenesis and enhances the thermogenic activity of offspring lambs by activating the AMPK-PGC1a signaling pathway.

Objective: To compare pregnancy outcomes between patients undergoing combined hysteroscopy and hysterosalpingo-contrast sonography (HyCoSy) versus hysteroscopy alone prior to intrauterine insemination, and to evaluate the safety and clinical value of the combined procedure in the diagnosis and treatment of infertility. Methods: A retrospective analysis was conducted on clinical data from 385 patients who underwent hysteroscopy at Peking University Third Hospital between October 1, 2020 and September 30, 2022, and subsequently received their first cycle of artificial insemination with donor sperm (AID) within six months. Pregnancy outcomes were compared between the group receiving combined hysteroscopy with four-dimensional HyCoSy (hysteroscopy+4D-HyCoSy group) and the group receiving hysteroscopy alone (hysteroscopy group). Multivariate logistic regression was used to analyze factors influencing pregnancy outcomes after AID. Results: Among the 385 patients included, 79 achieved clinical pregnancy. The clinical pregnancy rate (24.9%, 53/213) and live birth rate (21.1%, 45/213) in the hysteroscopy+4D-HyCoSy group were significantly higher than those in the hysteroscopy group [15.1% (26/172) and 12.8% (22/172), respectively; all P<0.05]. There was no significant difference in tubal patency between the two groups (P>0.05); however, the time interval from tubal patency assessment to intrauterine insemination was significantly longer in the hysteroscopy group compared to the hysteroscopy+4D-HyCoSy group (median: 4.0 vs 2.0 months; P<0.001). Multivariate analysis showed that double insemination significantly increased clinical pregnancy rate compared to single insemination (OR=2.42, 95%CI: 1.02-5.72; P=0.044). An interval exceeding 6 months between tubal patency assessment and intrauterine insemination was identified as a risk factor for reduced clinical pregnancy (OR=0.35, 95%CI: 0.14-0.92; P=0.047). Additionally, neither the time interval from hysteroscopy to intrauterine insemination nor hysteroscopic findings and pathological diagnoses had significant effects on clinical pregnancy rates (all P>0.05). Conclusions: The combination of hysteroscopy and HyCoSy provides a safe and efficient approach for fertility assessment in infertile patients and improves clinical pregnancy rate and live birth rate in intrauterine insemination cycles. Hysteroscopy is recommended for patients with suspected endometrial or intrauterine abnormalities. If no previous tubal patency assessment has been performed or the last assessment was more than six months prior, combined hysteroscopy and HyCoSy may be considered to enhance the likelihood of clinical pregnancy.

In 2025, it is our considered opinion, based on 40 years of experience in full-time andrology and reproductive sciences, that varicocele as a cause of male infertility remains largely unproven. While some meta-analyses suggest a marginal benefit in specific patient subsets, these findings are often debated regarding their clinical significance or methodological limitations. Couples with impaired semen parameters can be effectively treated by intrauterine insemination and, if necessary, by advanced assisted reproductive technologies like intracytoplasmic sperm injection (ICSI). With unproven benefits, an invasive nature, no clear explanation for the actual mechanism of causation, an unpredictable outcome, and the availability of simpler, better alternatives like IUI and ICSI, I feel varicocelectomy is the unkindest cut of all.

This study aimed to evaluate blood lactate, anion gap, and ionised calcium levels as potential diagnostic biomarkers in calves with atresia coli, and to identify possible predisposing factors such as breed, gender, age, method of conception, number of lactations, and births. The study included twenty-two calves with atresia coli and ten healthy controls, all aged 1–11 days (median, 3 days), brought to Erciyes University Veterinary Faculty from Kayseri and nearby provinces due to non-defecation and abdominal swelling. Prominent clinical findings among the 22 calves with atresia coli included abdominal distension in 90.9%, anorexia in 81.8%, and depressed general posture in 86.4%. Blood gas analysis revealed significantly elevated lactate and anion gap and decreased ionised calcium and pH in atresia coli calves compared to controls (p &lt; 0.05). Anion gap (&gt;14.05 mmol/L) and ionised calcium (&lt;1.205 mmol/L) demonstrated high diagnostic accuracy (AUC: 0.964 and 0.872, respectively), suggesting their potential as supportive biomarkers for early detection of atresia coli. The study data revealed that male gender, artificial insemination, and calves born from the third or subsequent pregnancies are statistically significant risk factors for the development of atresia coli. Atresia coli in calves is characterized by specific clinical signs and significant changes in blood gas parameters, such as elevated lactate and anion gap and reduced ionised calcium and pH. Early detection using these markers can improve diagnosis, and further studies should focus on prevention by addressing these risk factors.

Sperm cryopreservation is a critical technique for improving the efficacy of artificial insemination in sheep breeding programs. This study evaluated the effects of different extenders and packaging methods on post-thaw ram sperm quality, ultrastructure, and potential reproductive performance. Semen was collected from five healthy Ossimi rams and extended using three cryopreservation media: Tris-egg yolk (Tris-EY), Tris supplemented with 1% soybean lecithin (Tris-SBL), or Tris supplemented with 2 mM butylated hydroxytoluene (Tris-BHT). The extended semen was then packaged in either plastic straws or pellets and cryopreserved in liquid nitrogen. Post-thaw sperm quality was assessed by evaluating progressive motility, viability, and membrane integrity. Sperm ultrastructure was examined using transmission electron microscopy (TEM) and reproductive index. A two-way ANOVA analysis was used to study the effects of the extender and the method of packing. Cryopreservation using straws significantly improved post-thaw sperm quality compared to pellets, demonstrating higher progressive motility (p = 0.003), viability (p < 0.0001), and membrane integrity (p < 0.0001). However, neither the packaging method nor the extender type significantly affected the plasma membrane status, acrosome integrity, or mitochondrial, tail, and nuclear damage (p > 0.05). Ultrastructural analysis confirmed that straws, regardless of the extender used (Tris-EY, Tris-SBL, or Tris-BHT), effectively preserved sperm ultrastructure, including the nucleus, head, acrosome, plasma membrane, and mitochondria. The methods of packaged and extender types did not significantly affect the conception rate and litter size in sheep (p > 0.05). However, it is worth noting that Tris-SBL in straws had the highest conception rate (85.7%) and litter size (1.5 per ewe), while Tris-BHT in pellets had the lowest values (71.4% and 1.2, respectively). This study confirmed that plastic straw packaging significantly improved post-thaw sperm quality. While Tris-EY showed higher reproductive performance, further research is needed to establish statistical significance in pregnancy rates and litter size.

Amniotic fluid (AF) profiling provides a minimally invasive window into early fetal physiology. We characterized the AF metabolome from first trimester (Day 68) Holstein dairy heifers (n=45), considering fetal sex, conception method [in vitro fertilization (IVF) vs. artificial insemination (AI)], and eventual pregnancy outcome as key variables. Multivariate statistics uncovered differentially abundant metabolites for each comparison - including markers that preceded spontaneous abortion - independently of recipient age, weight, gestation length, or fetal genetics. Thereafter, a machine learning algorithm using panels of six metabolites accurately predicted fetal sex (AUROC=0.76; P=0.023) and pregnancy viability (AUROC=0.81; P=0.018), while corroborating conception method (AUROC=0.91; P=0.001). External validation using AF (Day 42) from an independent cohort of beef heifers (n=22) reproduced the fetal sex classifier with similarly high sensitivity and specificity (AUROC=0.85, P=0.029). These findings reveal metabolic signatures that forecast fetal sex and pregnancy viability, while confirming distinct metabolic imprints of assisted-conception modalities. These data lay the groundwork for next-generation AF prenatal diagnostics in veterinary and human obstetrics.

Abstract Background and objectives Lactation is one of the defining features of mammals, yet many humans struggle with breastfeeding. One reason for this is that humans are unique among mammals in the degree of learning and support that they require to breastfeed successfully. Despite this, we know little about how social learning impacts breastfeeding, particularly outside the influence of biomedical systems. Methodology Qualitative and systematic interviews were conducted with 128 Namibian women on infant feeding norms and practice. Structured statements were analyzed with Cultural Consensus Analysis to determine whether a single cultural model exists and identify variance in individual cultural competencies. Results Cultural consensus analysis revealed a single cultural model for breastfeeding, with strong and consistent norms and a significant role for social learning. Both learning and instinct were invoked in women’s responses, speaking to the necessary and expected role of intensive support in the early postpartum period. Women also noted steep learning curves and clear expectations about infant feeding, which led to universal breastfeeding success and clear paths for troubleshooting difficulties. Conclusions and implications The breastfeeding support that Himba mothers receive is part of the legacy of assisted reproduction in humans. However, the features of intensive teaching and learning shown here are lacking in western models of infant feeding and postpartum care. These data suggest that protracted breastfeeding difficulties may result from a mismatch between the evolved socioecology of breastfeeding and current norms and practices that hinder social learning and impair support pathways.

The article explores the current state, dynamics, and development prospects of the reproductive rights movement in European countries. Particular attention is paid to the forms of struggle by women and men for access to artificial insemination, abortion, and surrogacy.The contribution of public, medical, and feminist organizations to the advancement of these rights is examined, as well as the role of state policy, legislative initiatives, and international support. Examples are provided from France, Germany, Poland, Sweden, Denmark, theNetherlands, Austria, Finland, and also Ukraine. It is shown that under the conditions of the armed conflict in Ukraine, there arises a need to preserve reproductive material in case of injury or death of one of the partners, which has led to new legislative changes. The text includes a table comparing national approaches, charts of protest movement activity, and a flowchart ofjudicial protection of reproductive rights.

In modern animal husbandry, reproductive biotechnologies such as artificial insemination (AI) play a key role in increasing productivity and improving the genetic potential of farm animals. AI allows not only to use agricultural material issued by producers in a larger amount of the male animal as efficiently as possible, but also to reduce the risk of spreading sexually transmitted diseases. The success of AI is largely determined by the quality of the sperm used, which depends on the methods of processing and storing the ejaculate. Among the existing methods of sperm selection, centrifugation occupies one of the leading places. This method is based on the separation of the power components of the ejaculate under the influence of centrifugal. As an example of centrifugation, two main combinations were found: standard and colloidal centrifugation. Standard centrifugation is a single separation of the ejaculate components, with this centrifugation mode under transformations in accordance with the species characteristics of spermatozoa: 200-400 rpm for 5-12 min for rodents, 720 rpm for 5 min for dogs, 2400 rpm for 5 min for stallions, 5000 rpm for 5 min for bulls, 3000 rpm for 3 minutes for goats, 2400 rpm for 3 minutes for boars. Colloid centrifugation involves the use of special colloidal solutions (Percolltm, Isolate®, Porcicoll®, Bovicoll®, Equipure Bottom Layer®, PureSperm®) for more effective separation of spermatozoa formed with morphological and functional forms. Colloid centrifugation allows you to obtain higher quality ejaculate, since it can separate not only seminal plasma, but also defective, low-motility spermatozoa, as well as cells with damaged DNA.