Daniele Zambelli

Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa.

Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport and the different species. The localization of GLUTs after capacitation and acrosome reaction highlighted the possible changes in their distribution because of the different functional moment. Only in dog spermatozoa changes in GLUTs distribution were demonstrated; these changes could be related to the different metabolic needs and modifications occurring in the sperm cell.

Correlation between the age of the conceptus and various ultrasonographic measurements during the first 30 days of pregnancy in domestic cats (Felis catus).

We ultrasonographically evaluated the prenatal development in cats, from the early phases to Day 30 of pregnancy, subjecting a group of pregnant cats (n = 12) to a daily ultrasonographic exam. The ultrasonographic images allowed us to measure the minor diameter of the gestational sac and the crown-rump length of the embryo/fetus. Ten subjects underwent ovariohysterectomy at specific intervals during the pregnancy, with the aim of comparing the ultrasonographic data with real data; only two subjects brought their pregnancy to term. The earliest ultrasonographic observation of the gestational sac was on Day 10 after mating, while the embryo could be measured only beginning with Day 18. This study allowed to gather useful new data in order to clinically monitor the normal course of pregnancy in cats and to date the gestational age.

Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen

Oocyte preservation is still a challenge in the cat. The aim of this study was to evaluate the efficiency of oocyte vitrification in cryoloop in the domestic cat and to assess the embryonic development after IVF with cryopreserved semen. In vitro matured cat oocytes were vitrified in cryoloop after exposure to 10% ethylene glycol (EG, 0.9 M) in hepes synthetic oviductal fluid (HSOF) for 1 min, 20% EG (1.8M) in HSOF for 1 min, and 40% EG (3.6M), 10mg/ml Ficoll 70 and 0.3M sucrose in HSOF for 20s. Warmed oocytes were fertilized in vitro with frozen-thawed semen collected by electroejaculation and presumptive zygote were cultured in vitro for 10 days. Results showed that percentage of degenerated oocytes was higher (P<0.01), while cleavage rate and morulae blastocysts rate on day 6 were significantly lower (P<0.01) for vitrified oocytes than control. Blastocyst rate on day 8 was higher (P<0.01) for control oocytes than vitrified counterparts, and also developmental ability was higher (P<0.05) for non-vitrified oocytes, while the hatched blastocyst rate on day 10 was higher (P<0.05) for vitrified oocytes than control. In conclusion cat oocytes can be vitrified in cryoloop with a fairly good survival rate, cleavage rate and embryo development until pre-implantation stage.

COMPARATIVE IMMUNOLOCALIZATION OF HEAT SHOCK PROTEINS (HSP) -60, -70, -90 IN BOAR, STALLION, DOG AND CAT SPERMATOZOA

Heat shock proteins (Hsp)-60, -70 and -90 are important testis chaperones that fulfil several functions during sperm cell maturation. In post-meiotic cells, their expression may change or may be undetectable and in some species it may be evident in mature spermatozoa. The aims of this study were to verify whether Hsp60, -70 and -90 are present in the sperm, and to compare their localization in boar, stallion, cat and dog spermatozoa by immunofluorescence. Hsp-60 immunoreactivity was detected in sperm midpiece in all the species examined. In stallion sperm, Hsp70 signal was localized in the sub-equatorial band, whereas immunoreactivity was evident on the neck of dog spermatozoa and on both neck and sub-equatorial region of cat spermatozoa. In agreement with our previous observations, a triangular fluorescent signal in the equatorial segment of fresh boar sperm was detected. Hsp90 immunoreactivity was present in different portions of sperm tail: in the midpiece of both boar and cat spermatozoa and in the neck and throughout the tail in dog and stallion spermatozoa, respectively. When capacitation and acrosome reaction were induced in boar, stallion and dog spermatozoa, no changes in both Hsp60 and -90 were recorded by either Western blot or immunofluorescence. After induction of acrosome reaction, a Hsp70 redistribution in boar spermatozoa and an increased percentage of stallion spermatozoa showing the post-acrosomal signal were observed although no changes were recorded by Western blot; in dog spermatozoa, no changes in Hsp70 were found by Western blot and immunofluorescence after capacitation and acrosome reaction.

Ultrasound Aspects of Fetal and Extrafetal Structures in Pregnant Cats

Prenatal feline fetal growth and utero-placental development were ultrasonographically evaluated using an ultrasound scanner with a 10 MHz sector probe. Uterus, placenta, embryo, fetus and fetal membranes in 16 pregnant cats were monitored during the course of pregnancy; 13 subjects underwent an ovariectomy on specific days while three subjects went to term. Various anatomic structures, fixed in Carson-buffered formalin, were sectioned and then compared to ultrasound images. By ultrasound examination it is possible to evaluate every stage of the fetal development; the gestational chamber can be seen on the 10th and the embryo inside the chamber on the 14th day. By the 20th day it is possible to evaluate all the fetal membranes, and later it is possible to appreciate organs and structures such as the stomach, intestine, eyes (crystalline lens), kidneys and the cerebral choroid plexi, on the 30th, 40th, 50th, 39th and 40th day respectively. Based on our observations, it will be simpler to locate anomalies of development or pathologies during ultrasound examination of pregnant queens.

In vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P<0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P<0.01), while CT sperm contained more spermatozoa with tail abnormalities (P<0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P>0.05) between CT and EP sperm. Nevertheless, no difference (P>0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste.

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P>0.05). Sperm membrane integrity was positively affected (P<0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P>0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6h after thawing (P>0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P>0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.

Validation of a Model to Develop a Symptom Index for Benign Prostatic Hyperplasia in Dogs

Benign prostatic hyperplasia (BPH) is a spontaneous and age-related condition in humans and intact male dogs. A symptom index for BPH in men was created by the American Urological Association. In this study, it has been developed and statistically validated as a model to assign an objective score to canine BPH severity based on clinical signs observed and/or subjectively reported to the veterinarian by dog owners. The medical records of the Animal Reproduction Unit of University of Bologna (Italy) were used to select dogs with a clinical diagnosis of BPH. A data set was built up, and the animals were included in the statistical analysis as dependent variables. A score of 1-3 was assigned to the disease severity of each case based on signs annotated, graded using a scale ranging from 1 to 4. Signs of BHP were entered as predictors while disease severity as dependent variable to generate the predictive model. The model was finally used to re-classify each case of the data set, and the percentage of corrected predictions calculated. Overall, 373 subjects were entered in the model. Between them, 243, 107 and 23 animals have been represented based on medical records with a BPH severity score of 1, 2 and 3, respectively. The model correctly predicted the response variable in 97.3% of the cases. In this study, a BPH symptom index was created for the first time in dogs, which may be useful to standardize BPH severity with an objective score and to evaluate the necessity, the kind and the effectiveness of treatment.

Artificial neural networks for the definition of kinetic subpopulations in electroejaculated and epididymal spermatozoa in the domestic cat

This study was designed for the identification of different sperm kinetic subpopulations in feline semen using artificial neural networks (ANNs) and for the evaluation of the effect of ejaculation on motility patterns of these subpopulations. Seven tomcats presented for routine orchiectomy were electroejaculated, and after 5 days, orchiectomized and epididymal tail sperms were collected. Sperm motility characteristics were evaluated using a computer-assisted sperm analyzer that provided individual kinetic characteristics of each spermatozoon. A total of 23 400 spermatozoa for electroejaculated and 9200 for epididymal tail samples were evaluated using a multivariate approach, comprising principal component analysis and ANN classification. The multivariate approach allowed the identification and characterization of three different and well-defined sperm subpopulations. There were significant differences before (epididymal tail spermatozoa) and after (electroejaculated sperm) ejaculation in sperm kinetic subpopulation characteristics. In both epididymal and ejaculated samples, the majority of subpopulation was characterized by high velocity and progressiveness; however, the electroejaculated samples showed significantly higher values, suggesting that the microenvironment of the epididymal tail could affect the sperm motility or, alternatively, seminal plasma could increase the kinetic characteristics of the spermatozoa, indicating that only after ejaculation, the spermatozoa express their motility potential. Nevertheless, further studies are required to clarify the functional significance of each kinetic subpopulation.

Sex-sorted canine sperm cryopreservation: limits and procedural considerations.

The aim of this study was to define a protocol to store dog sperm before and after sorting to obtain an insemination dose sufficient to allow the conception by artificial insemination. Experiment 1 and 2 were performed to evaluate the more appropriate extender for preserving at room temperature dog sperm before and after sorting. Four extenders were tested: (1) Tris-fructose-citrate (TFC), (2) Tris-glucose-citrate (TGC), (3) modified Tyrodes albumin lactate pyruvate medium (mTALP), and (4) third fraction of the ejaculate (after centrifugation at 5000× g for 10 minutes; III FRAC). Experiment 3 and 4 were performed to evaluate the ability of dog semen to withstand sex sorting and freezing/thawing. Modified Tyrodes albumin lactate pyruvate medium was the best extender for canine sperm storage at room temperature (20 °C-25 °C) before (total motility: TFC, 8.3 ± 1.7; TGC, 50.0 ± 11.5; mTALP, 70.0 ± 0.1; III FRAC, 25.0 ± 1 0.4; P < 0.05) and after sorting (total motility: TFC, 7.3 ± 1.5; TGC, 10.3 ± 1.5; mTALP, 33.3 ± 6.7; III FRAC, 8.7 ± 5.8; P < 0.05), even if at 24-hour sorted sperm quality was impaired in all extenders tested herein. Sperm quality decreased after sorting (total motility: control, 92.5 ± 0.9; sorted, 52.9 ± 6.0; P < 0.05) and, especially, after freezing/thawing (total motility: frozen control, 25.7 ± 4.1; frozen sorted, 2.4 ± 1.2; P < 0.05). In conclusion, mTALP is an appropriate medium for canine sperm storage before and soon after sorting (hours), but a long storage period of sexed sperm at room temperature is not adequate. Cryopreservation greatly impaired sperm quality, and further studies are needed to optimize the freezing protocol for sexed dog sperm.