Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats

The effect of vaginal and cervical deposition of liquid semen stored at room temperature on the fertility of goats was tested in a field trial in which 217 Norwegian Dairy goats aged between 6 months and 7.5 years from 14 farms were inseminated after natural oestrous. Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return and kidding rates of 87.0 and 78.0%, and vaginal insemination gave 85.5 and 74.3%, respectively. There was no significant difference between the cervical and vaginal inseminations (P = 0.59 for the 25-day non-return and P = 0.40 for the kidding rates). Farm had a significant effect on the 25-day non-return rate (P = 0.03) but not on the kidding rate (P = 0.07). There were no significant differences between the fertility rates for different bucks (P = 0.36 for the 25-day non-return and P = 0.15 for the kidding rates). Fertility results after vaginal insemination were encouragingly high. Vaginal insemination is a simple, less costly and time consuming technique compared to others, also bringing into focus the animal welfare aspects of the artificial insemination procedure. As the final goal is to establish a technique that could be applied similarly on a large scale by all farmers, vaginal insemination must be considered as a method that would simplify the use of liquid buck semen in Norway.     

Danish A.I. field data with sexed semen

The objective of this study was to compare conception rates, non-return rates and sex ratios of sexed and conventional semen from the same sires in commercial dairy herds in Denmark. The semen was produced from three bulls from each of the three major dairy breeds in Denmark: Holstein, Jersey and Danish Red Dairy Breed (nine bulls total), in order to answer questions on breeds differences in field results. AI was performed by trained technicians using a minimum of 150 doses of sorted sperm and 50 control doses from each bull. During the trial, a total of 2087 doses were used in 63 herds.The trial showed that the conception rate using sorted semen was 5% points lower than with conventional doses for Danish Reds, 7% points for Jerseys, and 12% points for Holsteins. Translating this into non-return rate revealed differences of 10-20% points among bulls. These differences are thought to be a good indicator of what to expect from commercial use of sexed semen.The sex ratios varied from 89% to 93% female calves among breeds, which on average is consistent with the theoretical average sex ratio of 93% females considering the low number of inseminations.     

In vitro fertilization as a predictor of fertility from cervical insemination of sheep

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.     

Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa

This study analyzes the relationship between chromatin condensation and field fertility, expressed as 90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen-thawed semen from five bulls (six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%, were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No correlation was present between chromatin condensation and NRR. However, significant correlation was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm chromatin abnormalities and to detect animals of higher reproductive potential.     

Semen assessment, fertility and the selection of Hereford bulls for use in AI

Nineteen young Hereford bulls were used to study the relationship between semen characteristics and fertility in artificial insemination following 15 320 inseminations. Seven measures of sperm motility, morphological abnormalities, the release of hyaluronidase, ATP content and sperm head measurements were examined as predictors of fertility (49-day fixed-interval non-return rate). Two assessments of motility, three categories of abnormal spermatozoa, acrosomal changes and the release of hyaluronidase had predictive power. Multiple regression analysis showed that a combination of sperm motility after dilution in saline, motility after thawing and the proportion of coiled tails and proximal protoplasmic droplets provided the best prediction of fertility and allowed bulls to be ranked in order of observed non-return rate (%) with a Spearman correlation better than +0.80.     

Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract

Semen extenders containing components such as egg yolk and skim milk are difficult to standardize and they introduce the risk of microbial contamination. A well-defined extender not originating from animal tissues would present a valuable contribution to the AI industry. We evaluated the fertility of bovine semen cryopreserved with 3 different extenders: 1) TRIS-Standard, prepared at 2 local AI laboratories, containing 20% (v/v) pasteurized egg yolk, 2) TRIS-Concentrate, prepared by adding 20% (v/v) pasteurized egg yolk and 1:5 (v/v) nonpyrogenic water, and 3) Biociphos Plus, a soybean extract containing extender, prepared by adding 1:5 nonpyrogenic water. Ejaculates of 4 Holstein bulls were split into 3 aliquots and cryopreserved with the 3 extenders. Prior to this study, the semen dose-response curve for each of the 4 bulls was developed in a field trial by freezing the semen and randomly distributing the straws throughout the Netherlands for insemination. Optimal semen doses were thus established to detect the effect of extenders on fertility, evaluated by 56-day non-return rate (NR56), and by the estimated conception rate and the calving rate, given a conception. We used the multiphasic model developed by Grossman et al. (7). A total of 22,246 first and second inseminations were recorded. The NR56 ranged among bulls from 67.0 to 70.1% for Tris-Standard, from 67.5 to 69.9% for Tris-Concentrate and from 60.2 to 66.7% for Biociphos Plus. No significant differences in NR56 were detected between Tris-Standard and Tris-Concentrate (P=0.54), whereas Biociphos Plus resulted in a significantly lower NR56 than Tris-Standard and Tris-Concentrate (P<0.05). Estimated conception rate was 72.1, 73.6 and 69.6% and estimated calving rate, given a conception was 80.6, 78.3 and 77.1 for Tris-Standard, Tris-Concentrate and Biociphos Plus, respectively. These results indicate that 1) semen extended with a custom made TRIS-Concentrate can be succesfully used in the field resulting in comparable fertility with Tris-Standard; 2) semen extended with Biociphos Plus results in a significant reduction in the NR56; 3) extender source may affect both conception rate and calving rate, given a conception, i.e., extrinsic and intrinsic sperm factors (4).     

Refinement of two female fertility QTL using alternative phenotypes in French Holstein dairy cattle

Two quantitative trait loci (QTL) affecting female fertility were mapped in French dairy cattle. Phenotypes were non-return rates at 28, 56, 90 and 282 days after insemination. On chromosome 3, a QTL was significant at 1% for non-return rate at 90 days, suggesting that it affects early fertility events. An analysis of SLC35A3, which causes complex vertebral malformation, excluded this gene from the QTL interval. On chromosome 7, a QTL was almost significant (P = 0.05) for non-return rate at 282 days. This QTL was associated with abortion and stillbirth problems. Use of appropriate phenotypes appeared important for fine-mapping QTL associated with fertility.     

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57-78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r = 0.689, P < 0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r = 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility.     

The predictive value of routine semen evaluation and IVF technology for determining relative boar fertility

Practical techniques for assessing semen quality in order to predict male fertility are still needed. The principal objective of this experiment was to evaluate routine laboratory evaluation and in vitro fertilization (IVF) techniques as predictors of relative boar fertility using a low-dose AI protocol. Nine boars were evaluated during a 6.5+/-1 mo period, beginning at 29-32 wk of age. Ejaculates were evaluated for motility, morphology and concentration, diluted to 1.5 billion sperm in 50 mL extender, and used to breed 50+/-5 gilts over the same period. On nine occasions, a specific aliquot of the ejaculate's first sperm-rich fraction was evaluated using IVF procedures. Boars differed (P<0.001) consistently for pregnancy rate (from 73 to 98%), farrowing rate (71-98%) and total born (8.8-12.0). Routine semen evaluation and IVF parameters that presented significant differences between boars, but no differences in time and no boar by time interaction, were used to correlate in vivo fertility. A multiple regression model based on routine semen evaluation parameters accounted for up to 27 and 22% of the variation of fertility index and total piglets born, respectively, whereas male pronuclear formation rate was the IVF variable that accounted for 17 and 12% of the variation in farrowing rate and fertility index, respectively. Collectively, we inferred that the use of low sperm numbers for AI, determination of pregnancy rate at Day 30, motility of extended semen after 7 and 10d, and specific IVF parameters may be useful for identifying relatively infertile boars that are not currently excluded from use in existing commercial boar studs.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

Variability in relationships between semen quality and estimates of in vivo and in vitro fertility in boars

The present experiment was designed to characterize relationships between common semen quality and fertility estimates for three boars known to differ in farrowing rate, number of pigs born alive, and monospermic penetration rate. The approach chosen to accomplish this was to monitor semen quality from these boars and use their semen alternately for either artificial insemination or in vitro fertilization for 40 weeks. This strategy relied on the variability in semen quality parameters that normally occurs in an individual boar over time. When comparisons were made among boars, farrowing rates, numbers of pigs born alive, and monospermic penetration rates were significantly different, but progressive motility, normal head and tail morphology, and acrosome morphology were not. However, when comparisons were made among ejaculates within individual boars, there were significant effects of semen quality on both in vivo and in vitro fertility. For boar 3495, the proportion of spermatozoa exhibiting progressive motility and distribution of spermatozoa in a percoll gradient had a positive linear effect on number born alive and monospermic penetration rate, respectively. For boar 2901, quadratic equations best described changes in litter size as a function of progressive motility and normal acrosomes. In addition, monospermic penetration rate increased linearly as normal acrosomes and the proportion of spermatozoa recovered from a percoll gradient increased. For boar 4291, the relationship between progressive motility and number born alive and between normal acrosomes and number of pigs born alive were also quadratic. However, a significant linear relationship was present only between normal acrosomes and monospermic penetration rate. These results demonstrate that simply relying on the means of common semen quality estimates from some boars has limited value in terms of being used as a prospective indicator of their in vivo or in vitro fertility. In contrast, characterization of relationships between semen quality and fertility estimates is useful for estimating differences in the fertility of ejaculates from individual boars. However, both quantitative and qualitative differences in these relationships among boars are present and a given semen quality estimate that is a good predictor of in vivo or in vitro fertilization for one boar, may not be applicable for others.     

Fertility results after different thawing procedures for ram semen frozen in minitubes and mini straws

The effect of different thawing procedures for ram semen frozen in minitubes and mini straws on the fertility of sheep was tested in a field trial in which 727 Norwegian crossbred ewes, aged between six months and five-and-a-half years from nine farms, were inseminated with frozen-thawed semen in natural estrous. Minitubes were thawed at 70 degrees C for 8 s (T70) and mini straws either at 70 degrees C for 5 s (S70), 50 degrees C for 9 s (S50), or 35 degrees C for 12 s (S35). Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return rates of 78.7, 69.0, 73.6, and 72.9% (overall 73.6%), respectively, and lambing rates of 77.6, 66.1, 71.4, and 68.9% (overall 71.0%), respectively. There was a significantly higher lambing rate for T70 compared to S35 (P=0.03) and S70 (P=0.02), respectively, but not compared to S50 (P=0.29). Age of the ewes (P=0.02), farmers (P=0.02) and the interaction between farmer x straw type/thawing temperature (P=0.01) had a significant effect on the lambing rate. In conclusion, the superior fertility results achieved for minitubes compared to mini straws have to be carefully evaluated in relation to the possible application of a more rational semen production and simplified semen handling at AI, when using mini straws thawed at 35 degrees C.     

Relationship Between Testicular Measurements,Body Weight and Semen Quality in Young Dairy Bulls

Dairy bulls, 322 Ayrshires (Ay) and 85 Friesians (Fr), were studied at the age of 11 months. Of the bulls, 286 Ay-bulls and 80 Fr-bulls produced semen of acceptable quality for use in A.I. Scrotal circumference, tonometer measure, scrotal fold thickness, 1-year body weight and testicular palpation were used to predict unsuitable bulls for A.I. Non-return rate was used as a measure of fertility. Scrotal fold thickness and 1-year weight had no significant correlation with fertility or semen quality. Scrotal circumference had a significant positive correlation with fertility. Tonometer ratio had a significant negative correlation with fertility. Testicular palpation was the best basis for predicting bulls with poor semen quality in this study. Twelve bulls were recorded as having testicles of different sizes, 1 testicle being more than 20 % bigger than the other. Only 2 of these 12 bulls produced semen of acceptable quality. One of these 2 bulls was, after slaughter, diagnosed as having a hereditary testicle disease. Friesians were shown to have significantly higher fertility than Ayrshires.     

Effect of gonadotrophin releasing hormone on conception rate in repeat-breeder dairy cows

The effect of gonadotrophin releasing hormone (GnRH) on conception rate was studied in 961 repeat-breeder cows in five California dairy farms. Cows were injected with GnRH or saline at the time of the fourth insemination. All breeding was by artificial insemination (AI) using frozen semen from two bulls. Conception rate for GnRH-treated cows was higher than for controls (47.0 vs 37.7%, P < 0.01). There was no significant difference in conception rate between the two bulls or among five AI technicians. How GnRH acts upon fertility in repeat-breeder cattle has not been determined.     

Progesterone concentrations in milk fat at first insemination--effects on non-return and repeat-breeding

The relationship between milk fat progesterone concentration at first artificial insemination (AI) and reproductive performance of Norwegian Red Cattle dairy cows was investigated in a field study. Fifteen AI technicians collected milk samples from 2250 cows from 458 herds and progesterone was analysed in the milk fat portion of the samples. Logistic regression revealed decreased probability of non-return, and increased likelihood of repeat-breeding, with increasing progesterone concentrations at insemination. The odds ratio for non-return and for repeat-breeding between the minimum and maximum value for milk fat concentrations between 4 and 32.2ng/ml was 4.5 (P < 0.01) and 3.2 (P = 0.01), respectively. The variable 'Technician' did not significantly influence non-return rate or repeat-breeding. Progesterone concentration at AI was positively related to calving to last insemination interval, and to number of inseminations per cow (P < 0.05). The association between the progesterone concentration at insemination and calving interval was only marginally significant. Technician was significantly (P < 0.01) associated with interval from calving to first AI, interval from calving to last AI (P = 0.05), and number of AI per cow (P = 0.01). Technician was not significantly related to the calving interval.We conclude that increased suprabasal progesterone concentrations at the time of first insemination, and higher return rate at AI may, at least partly, be due to endocrinological asynchrony at AI, a condition which may lead to decreased fertility.     

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis

A precise estimation of the fertilizing ability of a boar ejaculate would be very useful to improve pig assisted reproduction results. For this purpose, we tested the mathematical combination of several parameters of the boar semen quality analysis, including the computer-assisted semen motility analysis (CASA), as a predictive fertility tool. The utilized mathematical relations among parameters were logistic and linear regressions. Two mathematical models obtained by logistic regression involving Osmotic Resistance Test (ORT Test), Hyperosmotic Resistance Test (HRT Test) and viability of fresh samples, showed a significant (P<0.05) correlation between semen characteristics and conception rate. However, none of the obtained models produced a significant correlation model between semen characteristics and prolificacy. The CASA analyses show that three separate subpopulations of spermatozoa with different motility characteristics coexist in boar ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among boars, but no clear relationship between motile subpopulation structure and fertility was obtained. Our results support the belief that the predictive use of the results obtained in a standard boar semen quality analysis can reasonably be achieved by applying logistic correlation analyses among several function parameters of boar semen quality analysis and in vivo conception rates obtained after artificial insemination (AI).     

Frozen bovine semen quality and bovine cervical mucus penetration test

Research has been carried out to test bovine cervical mucus penetration (penetration) as a means for evaluating frozen-thawed bovine semen. A commercially available cervical mucus penetration test kit (the kit) was used. A total of 158 previously frozen semen samples collected from 61 bulls were thawed in a 37 C water-bath for 2 minutes. Four ways to estimate penetration were compared using the distance traveled during 90 minutes 1) at 21 C, or 2) at 37 C, by 3) the first solitary mobile spermatozoon, or by 4) the front of the mass of the mobile spermatozoa. Penetration was measured using phase contrast microscopy and a millimeter grid. Spermatozoal quality parameters (concentration, total motility, progressive motility, acrosome integrity, total sperm integrity and cytoplasmic droplets) were measured and the correlation to penetration was calculated. The best way to assay penetration with the kit was by measuring the penetration of the first solitary mobile spermatozoon at 37 C. Semen quality variability was significant (P < 0.05) relative to penetration. Linear correlations between penetration and acrosome integrity r=0.42 as well as between penetration and total sperm integrity r=0.53 were highly significant (P < 0.001). There was significant linear multiple regression between penetration and acrosome integrity (expressed as percentage and number) and total sperm integrity (expressed as percentage and number) (r=0.62; F=23.5147; P<0.0001). There was a significant difference between the average progressive motility of samples with penetration > 20 mm and samples with penetration 20%), but it is not useful to define the fertility level of semen samples.     

What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?

Until now it has been unclear to what extent the reduced fertility with sexed semen in the dairy industry is caused by too few sperm per AI dose, or by the effect of flow cytometric sorting, which is the established procedure for sexing semen. Therefore, we evaluated the effects of low sperm numbers per dose with and without sorting on non-return rates after 56 days (NRR56); in addition, we evaluated the effects of bulls, in order to further optimize use of sexed semen.Based on results of using sexed semen from seven Holstein bulls, an overall numerical decline of 13.6% in NRR56 was observed (P < 0.05). About two-thirds of this decline (8.6%) was due to the low dose (P < 0.05), and a third (5.0%) due to the process of sorting (P < 0.05). The effect of low dosage and sorting differed among bulls. We observed a sex ratio of 91.6% females for sexed semen from the first 131 calves born.Currently the best way to increase fertility of sexed semen is by closely monitoring fertility so that the highest fertility bulls are used, and by improving farm animal management. However, to make substantial progress, more in depth studies are needed on the sexing technology, especially on aspects such as sorting procedures and sperm dosage.     

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.     

Computerized and Subjective Assessments of Post-thaw Motility of Semen from Finnish Ayrshire AI Bulls in Relation to Non-return Rates

A semen analyser (Lazymot), was used to evaluate post-thaw motilities in 296 batches of semen from 74 Ayrshire bulls used for artificial insemination (AI). Motility was also assessed subjectively. A significant correlation was observed between assessments of motility using the Lazymot analyser and the subjective assessments. There was no correlation between post-thaw motility assessments and non-return rates in relation to the batches examined, which met Finnish criteria for use in AI. This suggests that criteria for post-thaw semen motility should not be increased beyond the present requirement for 40% motile spermatozoa.