A multivariate model for the prediction of pregnancy following laparoscopic artificial insemination of sheep

Abstract

The causes of variation in the success of laparoscopic artificial insemination (AI) in sheep are not well understood. As such, this study incorporated the contributions of multiple male and female factors relevant to the success of AI into a comprehensive prediction model for pregnancy success. Data from Merino ewes (N = 30 254) including age, uterine tone (1; pale/flaccid-5; turgid/pink), intra-abdominal fat (1; little to no fat present-5; high fat), time of insemination and sire used, were recorded during AI. A subset of semen per sire (N = 388) was thawed and assessed for volume, subjective motility, sperm concentration, and morphology. Sperm motility (CASA), viability and acrosome integrity (FITC-PNA/PI), membrane fluidity (M540/Yo-Pro), mitochondrial superoxide production (Mitosox Red/Sytox Green), lipid peroxidation (Bodipy C11), level of intracellular reactive oxygen species (H₂DCFDA) and DNA fragmentation (Acridine Orange) were also assessed 0, 3 and 6 h post-thaw. Logistic binomial regression revealed sperm concentration (P < 0.001), CASA parameters at 0 h (PCA3; P = 0.03), viable acrosome intact sperm at 6 h (P = 0.02), abnormal morphology (P < 0.001), uterine tone (P < 0.001) and intra-abdominal fat (P = 0.03) of ewes influenced likelihood of pregnancy. Results generated will help standardise the pre-screening and selection of semen and ewes prior to artificial breeding programs, reducing variation in the success of sheep AI.

Keywords: Acrosome; Concentration; Intra-abdominal fat; Laparoscopic; Morphology; Motility; Sheep; Sperm; Uterine tone; Viability.

Fig. 1

Variation in pregnancy rates for each site (deidentified, N = 30) recorded during the 2021, 2022 and 2023 breeding seasons. Calculated as the proportion of ewes pregnant compared to the total number of ewes inseminated per site.

Fig. 2

Pregnancy rate for each sire (deidentified) throughout the three breeding seasons, n = 388. Calculated as the proportion of ewes pregnant compared to the total number of ewes inseminated per sire. The number of ewes inseminated per sire ranged from 7 to 361, with an average of 69.74 ± 2.15 ewes.

Fig. 3

The Odds Ratio plot shows the relationship between an increase in freezing concentration per pellet or straw on the predicted probability of a ewe falling pregnant if she was laparoscopically inseminated with that sample. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (shaded blue area). Black markers along the x-axis indicate the spread of raw data per individual sire within the model. The blue line indicates the odds ratio of the sample at the given freezing concentration.

Fig. 4

The Odds Ratio plot shows the relationship between an increase in the number of abnormal spermatozoa on the predicted probability of an ewe falling pregnant if she was laparoscopically inseminated with that sample. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (shaded blue area). Black markers along the x-axis indicate the spread of raw data per individual sire within the model. The blue line indicates the odds ratio of the sample at the given abnormal morphology percent.

Fig. 5

The Odds Ratio plot showing the effect of the percentage of Acrosome Intact and Viable spermatozoa on the predicted probability of an ewe falling pregnant if she was laparoscopically inseminated with that sample. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (shaded blue area). Black markers along the x-axis indicate the spread of raw data per individual sire within the model. The blue line indicates the odds ratio of the sample at the given percentage of viable spermatozoa with intact acrosomes.

Fig. 6

The Odds Ratio plot shows the relationship between the CASA PC3 at 0 h post-thaw on the predicted probability of an ewe falling pregnant if she was laparoscopically inseminated with that sample. The PC3 is centred around the mean value of 0, and each individual point is represented by its deviation (distance) from this mean, measured in standard deviations. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (shaded blue area). Black markers along the x-axis indicate the spread of each PC3 point calculated by the PCA. The blue line indicates the odds ratio of the sample at the given abnormal morphology percent.

Fig. 7

The Odds Ratio plot shows the relationship between the uterine tone groups on the predicted probability of an ewe falling pregnant if she was laparoscopically inseminated. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (pink error bars). The blue dots indicate the odds ratio of the ewe with the given uterine tone score.

Fig. 8

The Odds Ratio plot shows the relationship between the intra-abdominal fat groups on the predicted probability of an ewe falling pregnant if she was laparoscopically inseminated. The predicted probability was generated from the model in RStudio, with 95% Confidence Interval (pink error bars). The blue dots indicate the odds ratio of the ewe with the given intra-abdominal fat score.