Canine-chilled sperm: study of a semen extender made with low-density lipoproteins from hen egg yolk supplemented with glutamine.

The objective of this study was to evaluate the effects of a combination of 6% low-density lipoproteins (LDL) and 20 mm glutamine in comparison with other extenders used for the refrigeration of canine semen: Tris egg yolk (EY) 20% and 6% LDL. The percentages of mobile spermatozoa after 4 days storage in a domestic refrigerator at +4 °C were 53.1%, 44.2% and 52.2% for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders respectively for 100% of the dogs. After 7 days of storage, these percentages fell to 37.8%, 26.4% and 33.6% in the same extenders for 50% of the dogs. In vitro fertility tests were performed with all of the extenders following the mobility results. These tests were conducted on the day of sampling (D0), and 48 and 96 h after sampling. The results of the hypo-osmotic swelling test were 82.6%, 81.2% and 85.7% on D0, 75.2%, 74.1% and 78.5% on D2, and 70.8%, 71% and 76.1% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. For the FITC/pisum sativum agglutinin (PSA) test, the results were 81.5%, 70.2% and 84.8% on D0, 78.9%, 62.3% and 84.2% on D2, and 72.7%, 59.6% and 73.7% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. The acridine orange test was positive; in nearly 100% of cases, none of the spermatozoa had been denatured on D0, D2 and D4. The 6% LDL + 20 mm glutamine and the 6% LDL extenders are capable of preserving spermatozoa that have been stored in a domestic refrigerator at +4°C for at least 4 days. This means that the spermatozoa retain good cytoplasmic membrane integrity, had not capacitated and contained intact DNA in comparison with spermatozoa preserved in the egg yolk extender. The duration of storage is a very important consideration when faced with the problem of sending semen over ever-greater distances.

In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: preliminary results of artificial inseminations.

A semen extender made with low density lipoproteins (LDL) has been used instead of a standard extender that is already available on the market for the cryopreservation of bovine semen. However, in order to extend its use to artificial insemination centres, in vivo fertility studies were required. Semen was taken from three bulls and frozen-thawed in two extenders: the LDL extender and a standard Tris-egg-yolk (20%) extender used by AI centres. The quality of the semen was assessed prior to artificial insemination: motility was assessed using an image analyser (Computer Assisted Semen Analysis (Hamilton Thorne)), and the integrity of the plasma membrane was assessed using the hypo-osmotic test (HOS test). For the first time, gestations were obtained following the artificial insemination of cows in the field (n=193) with semen that had been frozen-thawed in the LDL extender. No significant difference (p>0.05) was detected between the success rates of AI between the semen that had been frozen-thawed in the LDL extender (59.2%) and the control extender, Tris-20% egg yolk (65.3%). In conclusion, the in vivo fertility of semen that has been frozen-thawed in the LDL extender is maintained since gestations are obtained following AI.

Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: preliminary results.

Glutamine has been used in the composition of semen extenders in several species, but never in the bull. The aim of our study is to demonstrate the cryoprotective role of glutamine for freezing bovine semen and to determine concentration of the latter to improve the motility and trajectory characteristics of spermatozoa. Three experiments were undertaken with 21 ejaculates from three different bulls. In the first experiment, glutamine was added to 40, 80, and 120 mM of basic medium (BM) which consisted of Tris+glycerol 6.4% (v/v). In the second experiment glutamine was added to the 8% low density lipoprotein (LDL) diluent at 40, 80, and 120 mM. In the third experiment, the best concentration of glutamine was determined; this was then added to the LDL extender at 10, 20, 30, and 40 mM. The semen was diluted then frozen in the different media. Motility parameters were assessed using an image analyser following thawing. Experiment 1 demonstrated that glutamine had a cryoprotective effect; at 40 mM it gave superior motility parameters to those obtained with the basic medium (p<0.05). Experiment 2 demonstrated that the combination of LDL-glutamine 40 mM and 80 mM did not improve motility and even deteriorated it in comparison with the glutamine-free LDL extender. Experiment 3 demonstrated that the addition of 10mM of glutamine to the LDL medium lead to a significant improvement (p<0.05) in the motility of bull spermatozoa and could be used to improve bovine semen extenders.

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: comparison to Triladyl and Bioxcell.

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl, Bioxcell and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurusxBos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 x 10(6), 60 x 10(6) and 20 x 10(6)sperm/mL, corresponding to 30 x 10(6), 15 x 10(6) and 5 x10(6) sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell and Triladyl (p<0.05), but no significant difference was observed between Triladyland Bioxcell. Therefore we can conclude that LDL extender could be used instead of Triladyl or Bioxcellat low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.