Feng-Pang Cheng

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca2+/Ca2+‐ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm‐rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrodes medium (HBT containing 0.1 mM Ca2+), without (control sample) and with 2.5 μM Ca2+‐ionophore (induced sample) and incubated for 60 min at 38°C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5‐ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer‐driven three‐step freeze protocol and subsequently thawed for 50 sec in a 37°C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein‐conjugated peanut agglutinin (PNA‐FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD‐1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca2+‐ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca2+‐ionophore treatment correlated very negatively (r = −0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca2+‐ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca2+‐ionophore treatment followed by simultaneous determination PNA‐FITC and EthD‐1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors. Mol. Reprod. Dev. 55:289–298, 2000.

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Characterisation of the progesterone receptor on canine spermatozoa

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone-bovine serum albumin-fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 +/- 4%), but this labelling was markedly reduced (33 +/- 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 +/- 10% (onset) to 59 +/- 7% by 5 h, where it plateaued. Progesterone (P 4 ) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 ug/mL P 4 corresponding to 10 +/- 5%, 16 +/- 9%, 23 +/- 7% and 30 +/- 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P 4 -induced AR (12 +/- 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P 4 -induced AR (10 microg/mL) in capacitated ejaculated spermatozoa from 19 +/- 6% to 11 +/- 4% (h151, 1 : 10) and 12 +/- 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P 4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

Detection of matrix metalloproteinase (MMP)-2 and MMP-9 in canine seminal plasma

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality.

Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our laboratory recently confirmed the first case of encephalitozoonosis in a rabbit in Taiwan; the prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being infected. This study surveys the seropositivity of E. cuniculi using carbon immunoassay (CIA) and enzyme-linked immunosorbent assay (ELISA). Serological examination of 171 rabbits using CIA and ELISA showed that 63.2% (108/171) and 67.8% (116/171) were seropositive against E. cuniculi, respectively. Thirteen of the 14 rabbits (92.9%) with neurological symptoms were seropositive. Except for gender, health status and location had a significant effect on E. cuniculi seropositivity (p<0.05). Adult rabbits aged older than 4 months exhibited significantly higher seropositivity for E. cuniculi than young rabbits (p<0.05). In conclusion, this study shows that E. cuniculi is present and widespread among healthy rabbits in Taiwan. Therefore, the fields of veterinary and human medicine in Taiwan should be aware of this zoonotic issue and the resulting public health concern of encephalitozoonosis.

The combinatorial effect of different Equex STM paste concentrations, cryoprotectants and the straw-freezing methods on the post-thaw boar semen quality.

This study was to evaluate the combinatorial effect (14 treatments, A-N) of different Equex STM paste concentrations, cryoprotectants and the straw-freezing method on the post-thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post-thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post-thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw-freezing method changes between glycerol and DMA.

Ovarian cycle of the captive formosan gem-faced civets (Paguma larvata taivana)

Formosan gem-faced civets are classified to be endemic sub-species of Paguma larvata in Taiwan. Little about their reproductive physiology has been reported. This study was designed to characterize the ovarian activity throughout the year and define ovarian cycle length and the lengths of its component phases. Serum samples were collected for enzyme immunoassay (progesterone and estradiol) from seven captive civets twice weekly for 1 year. Meanwhile, periodic changes in external genitalia (vulva swelling) and vaginal cytology were examined and recorded. Results showed estrous cycles exhibited two types: 18-day (18.5+/-1.1, n=64) and 28-day (27.6+/-1.0, n=28) as shown by progesterone and estradiol fluctuations and corresponding changes in vulva morphology and vaginal cytology. Both types showed a similar 7-day follicular phase, peaking progesterone at Day 7. The 18-day cycle type prevails in the spring and summer whereas the 28-day cycle type is significant in the autumn. In summary, female gem-faced civets are polyestrous (approximately 13 cycles/year), and non-typical seasonal breeders, with follicular phase and two distinct durations of luteal phases (diestrus) cycling throughout the year, but the frequency of ovarian cycles was remarkably gradually decreased from September to February of next year. Zoo Biol 0:1-11, 2007. (c) 2007 Wiley-Liss, Inc.

Complications during labour in a chihuahua due to diaphragmatic hernia

DIAPHRAGMATIC hernias complicating pregnancy or labour are rare in both veterinary and human medicine. Such cases are usually associated with a history of congenital or traumatic injury (Hill and Heller 1996, Genc and others 2003, Hamoudi and others 2004). In the veterinary literature, two cases of diaphragmatic hernia in dogs during pregnancy have been reported, but both lacked a detailed description (Sullivan and others 1969, Iwasaki and others 1999). This short communication describes a case of diaphragmatic hernia in a pregnant dog with no history of congenital defect or traumatic injury. A female four-year-old, short-coated chihuahua, at 64 days’ gestation of a second pregnancy, presented with sudden depression, tachypnoea with extension of the head and restlessness that had been observed for over two days. The pregnancy had been uneventful, and the dog’s previous pregnancy had resulted in three naturally delivered puppies. On admission, the dog appeared depressed and lethargic, and displayed laboured abdominal breathing and cyanosis. A dry, greenish residue of vaginal discharge was observed, with no sign of abdominal straining. Radiography identified loss of diaphragmatic line, loss of cardiac shadow and an intestinal gas shadow in the thorax (Fig 1). A diagnosis of diaphragmatic hernia was made. Three well-ossified fetuses were visible in the abdominal cavity. Ultrasonography showed that all of the fetuses were vital, with more than 150 heart beats per minute. As the dog was in the second stage of labour and exhibited clear respiratory distress, an immediate caesarean section was performed, with surgical repair of the diaphragm via laparotomy. During surgery, three fetuses with intact amniotic sacs were located in the uterine horns and removed. In addition, a lobe of liver, a partial portion of the duodenum and jejunum, and the pancreas were found to be herniated into the right hemithorax through a defect in the right hemidiaphragm (Fig 2). All herniated viscera were viable and free of any adhesion, damage or necrosis. They were reduced into the abdominal cavity and the defect was repaired. Postoperative recovery in an oxygen chamber was uneventful, and the puppies were returned to the dog when she was able to nurse. An uncomplicated recovery was confirmed when the dog was re-examined 14 days later. This case is the first recorded peripartum and surgical repair of a diaphragmatic hernia in a dog in which the patient had no previous congenital or traumatic injury. A high transdiaphragmatic pressure gradient is suggested as the major cause of this acquired herniation. The acute onset of dyspnoea and lack of any adhesion of the herniated viscera further indicates that the hernia was newly acquired rather than being of congenital aetiology. No matter what the cause, a diaphragmatic defect may remain ‘silent’ and only become symptomatic because of gravid uterus and increased intra-abdominal pressure during pregnancy, labour or in the immediate postpartum period (Hill and Heller 1996, Williams and others 2003). The management of a pregnant dog with symptomatic diaphragmatic hernia is challenging. Immediate surgical Veterinary Record (2007) 161, 103-104

Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality.

The current study aimed to investigate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen was collected from Duroc boars and was allocated to 32 treatment groups for cryopreservation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI) was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b) dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference observed among these three treatments. The farrowing rates after AI with post-thawed semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa.