Publications

Use of in vitro assessed semen quality criteria to predict fertility of bull semen
Storing fresh equine semen
Batellier, F., Duchamp, G., Vidament, M., Arnaud, G., Palmer, E., and Magistrini, M.
Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions.
Theriogenology. 50, 229-236.
1998

Abstract

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field.

Post-thaw viability of dog semen
Dobrinski, I., Lulai, C., Barth, A.D., and Post, K.
Effects of four different extenders and three different freezing rates on post-thaw viability of dog semen.
J. Reprod. Fertil. Suppl. 47:291-6., 291-296.
1993

Abstract

Ejaculates were collected from 17 dogs. After initial evaluation the ejaculates were split into four equal aliquots and diluted with either Triladyl, Pipes, IMV Universal or Tris-fructose-citric acid extender, all containing egg yolk and glycerol. The extended semen was frozen in 0.5 ml straws at a slow, intermediate or fast freezing rate. After thawing, the percentage of progressively motile spermatozoa and the velocity of forward motion was evaluated. There was no difference between the extenders with regard to progressive motility. The slow, intermediate and fast freezing rates resulted in the highest, intermediate and lowest motility values, respectively, regardless of extender. All four extenders were equally suitable for freezing canine semen at a slow freezing rate.

Fecundity of stored porcine semen
Kuster, C.E. and Althouse, G.C.
The fecundity of porcine semen stored for 2 to 6 days in Androhep and X-CELL extenders.
Theriogenology. 52, 365-376.
1999

Abstract

Extending the raw ejaculate prior to artificial insemination (AI) is beneficial, in part, due to the increased number of females that are bred from an ejaculate, along with prolonged shelf life of the semen. The objective of this study was to examine the affects of storage time on the fecundity of porcine semen diluted in 2 semen extenders, Androhep and X-CELL. A completely randomized design with a factorial arrangement of treatments was utilized in which 429 high quality, gel-free ejaculates from 48 boars were used in a timed, double insemination of 1,431 first-service gilts. The gilts were divided into groups and inseminated with semen stored in Androhep or X-CELL for 2 to 3 d, 3 to 4 d, 4 to 5 d, or 5 to 6 d prior to use (day of collection = Day 0). Sperm age was identical, and both extenders were used concurrently each day of the trial. Farrowing rate and litter size data were recorded. Farrowing rates did not differ between extenders through Days 4 to 5 of storage. Gilts inseminated with Androhep diluted stored semen showed a decrease (P < 0.001) in farrowing rate compared with those inseminated with semen extended in X-CELL stored for 5 to 6 d. Mean litter sizes did not differ between extenders through Days 2 to 3 of storage. Compared with the X-CELL extended semen, gilts inseminated with Androhep extended semen produced smaller litters when semen was stored for 4 to 5 d (P < 0.05). Within the Androhep treatment, smaller mean litter sizes (P < 0.05) were evident when the semen was stored for 3 to 4 and 4 to 5 d. No differences were detected in litter size or farrowing rate for gilts bred with semen stored for 2 to 6 d in the X-CELL extender (P > 0.1). The results of this study indicate that extender type influences the fertility potential of fresh porcine semen stored for 2 to 6 d. For optimal fecundity in gilts, semen extended with Androhep extender should be used for AI within 3 d. The X-CELL extended semen can be used for up to 6 d without significant decrease in litter size or farrowing rate. These recommendations are dependent upon using high quality semen that is properly handled from collection through insemination.

Improved viability following a storage period
Prathalingam, N.S., Holt, W.V., Revell, S.G., Jones, S., and Watson, P.F.
Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation.
Anim Reprod. Sci. 91, 11-22.
2006

Abstract

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 x 10(6) and 60 x 10(6) sperm/ml in a commercial long term extender (Eqcellsire; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 x 10(6) sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa (P < 0.01) and were less susceptible to osmotic stress (P < 0.01) than sperm diluted to 60 x 10(6) sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted (P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability (r2 = 0.98; P < 0.001) or osmotic resistance (r2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.

Bovine semen frozen in egg yolk
Bousseau, S., Brillard, J.P., Marguant-Le, G.B., Guerin, B., Camus, A., and Lechat, M.
Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents.
Theriogenology. 50, 699-706.
1998

Abstract

The addition of components of animal origin (egg yolk, milk) to most commercial diluents used to freeze bull semen represents a potential risk of contamination of the doses with bacteria or mycoplasma. A series of quantitative and qualitative analyses were performed to detect microbiological contamination observed in Biociphos plus (a new lecithin-glycerol based freezing salt buffer), in an egg yolk diluent (Triladyl) or in an egg yolk + milk-based (Laiciphos) diluent of bull semen. The 2 diluents containing animal products showed moderate (10 to 60 CFU/mL) contamination (17/17 samples) with bacteria or mycoplasma, or both, while no contamination was observed in the 6 examined batches of Biociphos plus. Biociphos plus was also compared with another commercial diluent (Laiciphos) for use in freezing bull semen intended for in vitro and/or in vivo fertilization. No difference (P > 0.05) could be detected between the 2 diluents for in vitro fertility rates (percentage of cleaved zygotes: 85.7% and 88.0%, respectively, for Laiciphos and Biociphos plus). Similarly, 2 series of comparisons conducted in dairy cows artificially inseminated with semen frozen in either Biociphos plus or Laiciphos showed no difference in fertilizing capacity (tested at 60 to 90 d; P > 0.05) irrespective of the age of the bulls (Trial 1, bulls aged 14 to 15 m.o.; Trial 2, bulls aged 2 to 5 yr, field trials). It is concluded that, in addition to maintaining the fertilizing capacity of bull semen at levels comparable to those observed with standard freezing diluent, Biociphos plus also prevents microbiological contamination by bacteria or mycoplasma, both of which are generally present in the various commercially available sources of egg yolk

Boar semen quality
Sutkeviciene, N., Andersson, M.A., Zilinskas, H., and Andersson, M.
Assessment of boar semen quality in relation to fertility with special reference to methanol stress.
2005

Abstract

The relationship between various semen evaluation tests and fertility in fertile and subfertile artificial insemination (AI) boars was examined. In total, 36 boars, 19 Finnish Landrace and 17 Yorkshire, were included. The average value of three ejaculates extended in an X-cell extender from each boar was used in the analysis. Based on nonreturn results (NR60d, later referred to nonreturn rate, NR%), the boars were divided into two groups: those with poor fertility (NR% < 80, n = 19) and those with normal or above average nonreturn rates (NR% = 83, n = 17). Semen quality was determined after 1 and 7 days of storage at 17 degrees C. Sperm motility before and after each methanol stress was assessed both subjectively and using a computer-assisted semen analyzer (CASA). The sperm cells were stained with calcein AM and propidium iodide and evaluated for plasma membrane integrity under an epifluorescence microscope. Propidium iodide and Hoechst 33258 dyes were used in parallel to stain sperm cells for fluorometric analysis with an automatic fluorometer. Sperm morphology was evaluated in stained smears. The percentage of sows reported as not having returned to estrus within 60 days after AI (nonreturn rate, NR%) and litter size of primiparous and multiparous farrowings were used as measures of fertility. Of the parameters analyzed, only CASA-assessed total sperm motility and methanol-stressed total sperm motility correlated significantly (P < 0.05) with nonreturn rate. Those tests presenting the highest correlation with nonreturn rate were CASA-assessed total motility (r = 0.54, P < 0.01) and subjective sperm motility (r = 0.52, P < 0.01) after 7 days of storage. The highest correlation with fertility at 1 day of storage was shown by methanol-stressed total sperm motility assessed with the CASA (r = 0.46, P < 0.01). The only semen parameter that correlated significantly (r = 0.37, P < 0.05) with litter size of multiparous farrowings was viability of seven-day stored semen stained with Hoechst 33258 and analyzed with a fluorometer. The methanol stress test described here could serve as a rapid test whose results could be used to predict NR% better than motility.

Intrauterine insemination of sows
Watson, P.F. and Behan, J.R.
Intrauterine insemination of sows with reduced sperm numbers: results of a commercially based field trial.
Theriogenology. 57, 1683-1693.
2002

Abstract

Artificial insemination (AI) in pigs requires 2-3 billion spermatozoa to achieve consistently high fertility with current practice of inseminating into the posterior region of the cervix. We have investigated the potential advantages of inseminating through the cervix into the caudal region of the uterus using lower sperm numbers. Total sperm doses from 22 boars of 3, 2 or 1 billion spermatozoa were packaged in 80 ml volumes in X-Cell extender in gene-flat-pack (Cochette) bags. A novel inseminating device, the Deepgoldenpig, was employed which permits the ready introduction of a narrow catheter through the cervix into the uterus without traumatic injury to the mucosa. This was compared with the standard Goldenpig device that allows semen to be deposited in the posterior folds of the cervix. Sows of two different genotypes and of parities ranging from 2 to 11 were used. They were selected solely on the basis of a weaning to estrus interval of 4-6 days. Two inseminations, with a 24 h interval between them, were carried out on each sow. Pregnancy was determined at 35 days by ultrasound scan, and farrowing and litter size recorded. Pregnancy and farrowing data were very similar. The standard inseminating device produced farrowing rates of 91.1, 91.8 and 65.8% for insemination with 3, 2 and 1 billion spermatozoa, whereas the deep insemination device gave rates of 90.5, 90.5 and 86.9%. Only the 1 billion dose with the standard device was significantly different from the high dose control (P < 0.001). Similarly, the mean litter sizes with the standard device were 12.5, 12.6 and 10.3 and with the deep insemination device 12.3, 12.3 and 12.1. Only the 1 billion dose with the standard device was significantly lower (P < 0.001). None of the covariates differed significantly and there were no significant interactions with treatment. We conclude that transcervical insemination in the sow is simple, effective and safe, and allows the sperm dose to be reduced to 1 billion spermatozoa.

Five different vitrification devices
Camus, A., Clairaz, Ph., Ersham, A., Van Kappel, A.L., Savic, G., and Staub, C.
The comparison of the process of five different vitrification devices.
Gynecol. Obstet. Fertil. 34, 737-745.
2006

Abstract

Over the last decade, several methods have been designed to improve the survival rate of vitrificated embryos. Although some teams have succeeded, the main remaining drawback of these methods is that they do not provide a leak proof environment for cryopreserved biological samples. To respond to that demand in respect with the European reglementation, the Cryo Bio System company (CBS) designed the HSV High Security Vitrification Kit (HSV). This system is composed of three distinct parts, a High Security thermal-autogenic sealed clear straw, a capillary with its extremity in form of a gutter, and an introducer that can be mounted on the manipulation rod before introduction into the straw. In this study, we confirmed that the CBS vitrification kit is a suitable method for vitrification in association with a small amount of cryoprotector enriched viscous media such as 25 microM Ficool 400, 750 mM Sucrose, 1% Bovine albumin, 20% Dimethyl Sulfoxide and 20% Ethylene glycol in a Phosphate buffered saline solution. We also evaluated the speed of the temperature decrease during vitrification in comparison with four other commercially available non-aseptic methods and showed the protective role of the CBS system during transfer. These physical data have now been confirmed biologically by P. Vanderzwalmen who obtained easily reproductible good results with human embryo using our method. Today, the HSV represents the unique aseptic alternative device (EC and FDA approved) for embryos, oocytes, and biological samples vitrification.

Chilled and frozen semen
Decuadro-Hansen, G.
Chilled and frozen semen: the animal experience.
Gynecol. Obstet. Fertil. 32, 887-893.
2004

Abstract

The aim of this paper is to describe technologies that are currently being used in animal reproduction and those that will be used in the near future. The significant changes (news methods of conservation of chilled and frozen semen) operated these last ten years in connection with the development of animal insemination are all detailed. All these techniques are experiencing rapid improvements in terms of success rate.

Cushioned centrifugation technique
Ecot, P., Decuadro-Hansen, G., Delhomme, G., and Vidament, M.
Evaluation of a cushioned centrifugation technique for processing equine semen for freezing.
Anim Reprod. Sci. 89, 245-248.
2005
Post-thaw sperm parameters in ram semen
Gil, J., Lundeheim, N. Soderquist, L., and Rodriiuez-Martinez, H.
Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen.
Theriogenology. 59, 1241-1255.
2003

Abstract

Ejaculates were collected from 17 dogs. After initial evaluation the ejaculates were split into four equal aliquots and diluted with either Triladyl, Pipes, IMV Universal or Tris-fructose-citric acid extender, all containing egg yolk and glycerol. The extended semen was frozen in 0.5 ml straws at a slow, intermediate or fast freezing rate. After thawing, the percentage of progressively motile spermatozoa and the velocity of forward motion was evaluated. There was no difference between the extenders with regard to progressive motility. The slow, intermediate and fast freezing rates resulted in the highest, intermediate and lowest motility values, respectively, regardless of extender. All four extenders were equally suitable for freezing canine semen at a slow freezing rate.

Fertility of ram semen frozen in Bioxcell
Gil, J., Rodriguez-Irazoqui, M., Lundeheim, N., Soderquist, L., and Rodriguez-Martinez, H.
Fertility of ram semen frozen in Bioxcell and used for cervical artificial insemination.
Theriogenology. 59, 1157-1170.
2003

Abstract

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioxcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioxcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioxcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioxcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioxcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioxcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioxcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.

Motility of porcine spermatozoa
Vyt, P., Maes, D., Rijsselaere, T., Dejonckheere, E., Castryck, F., and Van, S.A.
Motility assessment of porcine spermatozoa: a comparison of methods.
Reprod. Domest. Anim. 39, 447-453.
2004

Abstract

Although widely used in practice, visual motility assessment of boar spermatozoa is a very subjective method. The aim of this study was to compare the visual motility assessment of boar spermatozoa with two objective, automated systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton-Thorne computer-based semen analyzer (HTR). In addition, concentrations as determined by the Burker counting chamber and HTR were compared. Motility of 30 semen samples from 30 different boars (22 Pietrain, seven Landrace boars and one Large White) was examined during three consecutive days, subjectively by two independent persons (visual motility assessment) and objectively with both automated systems. The use of the SQA-IIC and HTR for assessing boar sperm motility was evaluated and the repeatability of the measurements was estimated. The Sperm Motility Index (SMI), determined by SQA-IIC, and the percentage motile spermatozoa determined by the HTR showed a good correlation (r=0.71; p <0.05). The visual examination performed by Person 2 showed a good correlation with the SMI (r=0.81) and with the percentage of motile spermatozoa measured by the HTR (r=0.66) (p <0.05). There was a very poor correlation and a limited agreement between the visual assessments of both persons emphasizing the subjectivity of visual motility assessment. Nevertheless, motility scores of each person during the three consecutive days were highly correlated (r=0.67 and 0.72, p <0.05). The limits of agreement plots showed poor agreement between both persons and the HTR. The repeatability of measurements for most parameters evaluated by the HTR and by the SQA-IIC was good with coefficients of variation below 10%. In addition, for fertile Pietrain boars (n=22), reference values for the different HTR-parameters are presented showing a high curvilinear velocity (157.3 +/- 19.5 microm/s) and a low straightness and linearity of the movement of the spermatozoa (62.7 +/- 8.7 and 35.5 +/- 7.6%, respectively). Concentration as determined by the Burker counting chamber (56.0 +/- 16.8 x 10(6)/ml) was significantly higher compared with HTR measurement (37.6 +/- 7.7 x 10(6)/ml). The high number of counted cells and the low variation render the HTR concentration measurement more reliable. It can be concluded that visual motility assessment is highly subjective and should therefore be replaced by automated systems that allow for a more objective and detailed motility assessment of boar spermatozoa. In addition, based on the present results, highly repeatable results were obtained by the SQA-IIC and especially by the HTR.

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