Tom Kroetsch

Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen

A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.

Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

In vitro fertilization of oocytes by 37-year-old cryopreserved bovine spermatozoa

Abstract Bull spermatozoa that had been frozen in 1957 was tested for fertility after 37 years in storage. Two specimens of this “vintage” spermatozoa exhibited normal motility when thawed and successfully fertilized bovine oocytes in vitro. Of 670 oocytes incubated with vintage spermatozoa, 149 (22%) cleaved into 2-cell embryos, of which 32 (5% of the total number) developed into normal blastocysts.

Comparison of different methods for assessment of sperm concentration and membrane integrity with bull semen.

Assessing semen quality is crucially important for the exploitation of genetically superior sires in an artificial insemination (AI) program. In this study, we compare modern and conventional techniques to estimate bovine sperm concentration and membrane integrity. First, the NucleoCounter SP-100 was validated for sperm concentration and provided statistically reliable and repeatable estimates among aliquots and replicates of 25 fresh ejaculates. Sperm concentrations in 78 ejaculates were then determined with hemacytometer, flow cytometer, and NucleoCounter SP-100 and were significantly correlated (P < .001), with regression coefficients among these 3 techniques close to 1 (P < .01). However, the sperm concentration determined by hemacytometer was lower (P < .01) than by flow cytometer and NucleoCounter SP-100. Forty frozen-thawed semen samples were then assessed for sperm concentration and membrane integrity with hemacytometer, flow cytometer and NucleoCounter SP-100. Significant relationships were found for sperm concentration determined by hemacytometer and NucleoCounter SP-100 and for sperm membrane integrity determined by flow cytometer and NucleoCounter SP-100 (P < .01). Finally, the standard curves of sperm concentrations in 6 spectrophotometers, comparing optical density against counts drawn by hemacytometer and NucleoCounter SP-100 (n = 94 fresh ejaculates) showed different (P < .01) intercepts and regression coefficients (linear, quadratic, cubic). It was calculated that a breeding station can improve its production potential by 13% with the use of NucleoCounter SP-100 instead of hemacytometer for calibration of spectrophotometers. Flow cytometer and NucleoCounter SP-100 can be used with equal confidence to estimate sperm concentration and membrane integrity in domestic animals and human semen.

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls.

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = -0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.

Fertility-associated metabolites in bull seminal plasma and blood serum: 1H nuclear magnetic resonance analysis.

Early estimation of bull fertility is highly desirable for the conservation of male genetics of endangered species and for the exploitation of genetically superior sires in artificial insemination programs. The present work was conducted as a proof‐of‐principle study to identify fertility‐associated metabolites in dairy bull seminal plasma and blood serum using proton nuclear magnetic resonance (1H NMR). Semen and blood samples were collected from high‐ and low‐fertility breeding bulls (n = 5 each), stationed at Semex, Guelph, Canada. NMR spectra of serum and seminal plasma were recorded at a resonance frequency of 500.13 MHz on a Bruker Avance‐500 spectrometer equipped with an inverse triple resonance probe (TXI, 5 mm). Spectra were phased manually, baseline corrected, and calibrated against 3‐(trimethylsilyl) propionic‐2,2,3,3‐d4 acid at 0.0 parts per million (ppm). Spectra were converted to an appropriate format for analysis using Prometab software running within MATLAB. Principal component analysis was used to examine intrinsic variation in the NMR data set, and to identify trends and to exclude outliers. Partial least square‐discriminant analysis was performed to identify the significant features between fertility groups. The fertility‐associated metabolites with variable importance in projections (VIP) scores >2 were citrate (2.50 ppm), tryptamine/taurine (3.34–3.38 ppm), isoleucine (0.74 ppm), and leucine (0.78 ppm) in the seminal plasma; and isoleucine (1.14 ppm), asparagine (2.90–2.94 ppm), glycogen (3.98 ppm), and citrulline (1.54 ppm) in the serum. These metabolites showed identifiable peaks, and thus can be used as biomarkers of fertility in breeding bulls. Mol. Reprod. Dev. 82: 123–131, 2015.

Relationship between sperm apoptosis and bull fertility: in vivo and in vitro studies

The objectives of this study were to confirm the relationship of apoptosis-associated membrane and nuclear changes in bull spermatozoa with field fertility, to predict the fertility of beef bulls used for natural breeding and to study the role of DNA-nicked spermatozoa in early embryonic development. In Experiment 1, the relationship between fertility and different sperm populations identified by the Annexin V/propidium iodide (PI) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays was determined. Bull fertility was related to live (PPPin vitro cleavage and blastocyst rates was evaluated, using 30000 or 300000 spermatozoa per droplet. Cleavage rate was adversely affected (PP<0.05) in high DNA-nicked spermatozoa at the lower sperm concentration. In conclusion, the incidence of DNA-nicked spermatozoa is a useful marker to predict a bulls fertility potential. DNA-nicked spermatozoa showed adverse effects on early embryonic development.

Semen characteristics of genetically identical quadruplet bulls.

Modern cloning methods have become an important technology in artificial insemination which is used to create and maintain pools of genetically superior bull semen. In this study, semen from four identical quadruplet bulls (Q(1), Q(2), Q(3), and Q(4)) produced by blastomere separation was analyzed to evaluate the differences in reproductive potential, if any, that existed between the identical quadruplet siblings. Analysis of fresh semen collected from 1994 to 1996, showed lower progressive motility and lower sperm concentration for one bull (Q(3)) compared to his identical brothers (P<0.05). Semen characteristics following freezing-thawing procedures have also been tested for these quadruplet bulls. The percentage of motility, progressive motility, and mean path velocity were lower in Q(4) compared with Q(1). Moreover, intracellular calcium level and P25b level (P25b is a sperm surface protein proposed to be a potential bull fertility marker) were lower in Q(4) compared with his siblings (P<0.05). Cryodamage to Q(4)s frozen-thawed spermatozoa were confirmed by a lower percentage of embryo development after in vitro fertilization. Thus, the higher instability of cryopreserved spermatozoa from Q(4) and the lower semen production of Q(3), compared to their siblings, indicate that differences in semen characteristics can indeed exist among genetically identical animals produced by blastomere separation.

Highly dynamic temporal changes of TSPY gene copy number in aging bulls

The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.

In vivo and in vitro ageing results in accumulation of de novo copy number variations in bulls

We have identified de novo copy number variations (CNVs) generated in bulls as they age. Blood samples from eight bulls were collected and SNP arrayed in a prospective design over 30 months allowing us to differentiate de novo CNVs from constant CNVs that are present throughout the sampling period. Quite remarkably, the total number of CNVs doubled over the 30-month period, as we observed an almost equal number of de novo and constant CNVs (107 and 111, respectively, i.e. 49% and 51%). Twice as many de novo CNVs emerged during the second half of the sampling schedule as in the first part. It suggests a dynamic generation of de novo CNVs in the bovine genome that becomes more frequent as the age of the animal progresses. In a second experiment de novo CNVs were detected through in vitro ageing of bovine fibroblasts by sampling passage #5, #15 and #25. De novo CNVs also became more frequent, but the proportion of them was only ~25% of the total number of CNVs (21 out of 85). Temporal generation of de novo CNVs resulted in increasing genome coverage. Genes and quantitative trait loci overlapping de novo CNVs were further investigated for ageing related functions.