Cooling management effects on dry matter intake, metabolic hormones levels and welfare parameters in dairy cows during heat stress.

This research paper addresses the hypothesis that intensive cooling management during the summer improves the secretion of metabolic hormones in dairy cows. To test this hypothesis, we characterized the effect of different cooling managements on the different ghrelin isoforms and leptin secretion of 20 Israeli-Holstein dairy cows during 5 weeks during heat stress. The cows were divided into two groups: one was exposed to 5 cooling sessions per day (5 CS) and the other to 8 cooling sessions per day (8 CS). Blood was collected and leptin and ghrelin isoforms level were radioimmunoassayed. Analysis of the interaction between coolings and the week of the experiment showed that the 8 CS group consumed more food and produced more milk, although neither difference was statistically significant. In addition, the 8 CS group exhibited higher blood levels of acyl-ghrelin and leptin as compared to the 5 CS group. Conversely, the blood levels of total ghrelin were lower in the cows exposed to 8 CS as compared to cows from the 5 CS treatment. Furthermore, a significant correlation was found only between total ghrelin levels and the weeks, but not with other parameters examined. We further compared digestibility as well as stress parameters between the groups. We found that the 8 CS group cows ruminated and lay down more hours during a day and simultaneously had better activity time. No significant difference was detected between groups in milk yield and digestibility parameters. Our results suggest that intensive cooling management during the hot season influences the levels of metabolic hormones in the circulation and helps to mitigate the detrimental effect of heat stress on dairy cow welfare and production.

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

Double freezing of bovine semen.

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3h incubation at 37 degrees C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P<0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P< or =0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P<0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P=0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae).

Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67+/-11.67% (mean+/-S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49+/-3.24%, intact acrosome was detected in 63.74+/-2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups-highly active and progressively motile spermatozoa with SMI of 62.75+/-0.38 and low activity and poorly progressive with SMI of 46.16+/-1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.

New trends in gamete's cryopreservation.

We developed new techniques to improve freezing and vitrification of sperm, oocytes and embryos. Our novel freezing technology is based on 'Multi-Thermal-Gradient' (MTG) freezing that is used for sperm. The freezing apparatus has the ability to control ice crystals propagation by changing thermal gradient or the liquid-ice interface velocity which optimizes ice crystals morphology during freezing of cells and tissue. Using this apparatus we were able to freeze bull, stallion, boar, ram, fowl and human sperm with normal post-thaw motility/pre-freezing motility of 70-100%. The vitrification method includes the cooling of nanoliter sample (the 'Minimum Drop Size' technique) in 'super-cooled' liquid nitrogen (-210 degrees C), which maximized cooling rate to the highest physically possible (24-130000 degrees C/min). Using this method we achieved very high survival of bovine oocytes and embryos. Vitrification of oocytes at the MII stage resulted with cleavage and blastocyst rate of 50 and 20%, respectively. The vitrification of in-vitro production (IVP) of bovine embryos allowed the production of a healthy calf after embryo-transfer carrying the name 'Zegugit' (in Hebrew: made from glass).

Successful pregnancies in cows following double freezing of a large volume of semen.

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.