Occurrence of dermatomycosis (ringworm) due to Trichophyton verrucosum in dairy calves and its spread to animal attendants.

Persistent dermatomycosis (ringworm) caused by Trichophyton verrucosum affected 20 dairy calves aged between 3 months and 1 year and housed together. The infection also spread to 2 animal attendants working among the calves. The major clinical lesions observed on the affected calves were extensive alopecia and/or circumscribed thick hairless skin patches affecting the head, neck, flanks and limbs. The observed lesions persisted for more than 17 weeks and most of the calves did not respond to topical treatment with various anti-fungal drugs within the anticipated period of 9 weeks. Two animal attendants developed skin lesions that were circumscribed and itchy and there was good response to treatment following the application of anti-fungal skin ointment. Although ringworm in dairy animals in Kenya has not previously been associated with spread to humans, the potential is evident from this report.

In vivo fertilizing capacity of deep frozen boar semen packaged in plastic bags and maxi-straws.

Pooled ejaculates from six fertile boars were frozen under controlled conditions in Teflon FEP-film plastic bags (5 ml) and maxi-straws (2.5 ml) using 3% glycerol as cryoprotectant. The percentages of both post-thaw motility and normal apical ridges were significantly higher (P less than 0.001) for the bags (54.5 and 75%) than for the maxi-straws (40.1 and 59.4%) respectively. For evaluation of the in vivo fertilizing capacity of the frozen-thawed spermatozoa, 26 gilts were inseminated once 24 h after the first observation of standing reflex in their second oestrus, with 5 ml of semen (containing 5 billion spermatozoa) reconstituted in 80 ml of BTS from either bags or maxi-straws. Ova were recovered from the oviducts/uteri 2-4 days following insemination and examined for cleavage and sperm binding to the zona pellucida (ZP). Significantly higher rates (P less than 0.02) of fertilized ova were found in the bag-inseminated (75%) than in maxi-straw inseminated gilts (63%); and similarly their ova had significantly more spermatozoa in the ZP, irrespective of whether they were fertilized or nonfertilized. This study confirmed that the plastic bags are suitable and may be used for packaging single insemination doses of deep frozen boar semen for routine A.I. work.

Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.

Cryopreservation of boar semen. I: A literature review.

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.

Cryopreservation of boar semen. III: Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional freezing and thawing.

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.

Cryopreservation of boar semen in mini- and maxi-straws.

Split ejaculates from four boars were frozen with a programmable freezing machine, in mini- (0.25 ml) and maxi- (5 ml) plastic straws with an extender at either acidic (6.3) or alkaline (7.4) pH. Glycerol (3%) was used as cryoprotectant. The freezing of the semen was monitored by way of thermocouples placed in the straws. Post-thaw motility and acrosome integrity were evaluated; the latter using phase contrast microscopy, eosin-nigrosin stain and electron microscopy. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws than in maxi-straws. For the mini-straws, the motility was better when semen was exposed to an acidic environment during freezing, but this beneficial effect of the low extracellular pH was not evident when maxi-straws were thawed. The motility of the spermatozoa diminished significantly during the thermoresistance test (0 h and 2 h time) at 37 degrees C in a similar way for both straws and extracellular pH's. The freezing procedure, no matter the extracellular pH, did not cause major acrosomal damages, but significantly more normal apical ridges were present in the mini-straws than in the maxi-straws. This in vitro evaluation indicated that the freezing method employed was better for mini- than for maxi-straws since the freezing of the 5 ml volumes was not homogeneous, due to the large section area between the surface and the core of the straw.