Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15-60 degrees C/min) gives rise to best sperm survivals after freezing-thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks.

Twenty-two Serrana goats were studied through two successive estrous cycles in order to characterize their follicular dynamics during the breeding season. The ovaries of the goats were scanned daily by realtime ultrasonography and all follicles ≥3 mm were measured and classified. The data were classified by the number of follicular waves per goat to test the hypothesis that temporal and morphological differences between the last follicular wave of an ovary, irrespective of ovulation, will affect the selection of the next ovulatory wave.The mean interovulatory interval was 20.7 ± 1.0 days (mean ± S.D.). Three to five waves per estrous cycle were observed and 61.3% (19/31) of cycles had four waves. In estrous cycles with four waves, the day of onset of the first, second, third and fourth wave was 1.4 ± 1.0, 6.9 ± 1.4, 11.6 ± 1.8 and 16.8 ± 1.6, respectively. No differences (P > 0.05) were found between the day of onset of the first and second waves for estrous cycles with three, four or five waves. However, the day of onset of the third and fourth waves occurred later when the number of waves per estrous cycle increased (P < 0.001). The duration of the interwave interval (time between the day of onset of two consecutive waves) was longer when the second wave was ovulatory. The length of the growth phase (2.4 ± 0.9 days) and size (5.9 ± 0.7 mm) of the dominant follicle in the second wave were lower (P < 0.01) than for the first wave (3.3 ± 1.2 days and 6.6 ± 0.9 mm, respectively) and the fifth wave (4.1 ± 1.2 days and 7.5 ± 1.0 mm, respectively). Within pairs of ovaries, the onset of the last wave occurred later (P < 0.05) and was less variable in ovulatory ovaries (day 16.8 ± 1.4, n = 20) than in anovulatory ovaries (day 15.1 ± 3.7, n = 20). The length of the growing phase was longer (P < 0.001) in the last waves of ovulatory ovaries (3.1 ± 0.9 days) than in the last waves of anovulatory ovaries (1.7 ± 0.8 days). These results support the hypothesis that the day of onset of the ovulatory wave is related to or, at least, conditioned by the luteolysis and the decrease in plasma progesterone. * Corresponding author. Tel.: +35 1259350417; fax: +35 1259350480. J. Simões et al. / Animal Reproduction Science 95 (2006) 16-26 17 In summary, the estrous cycle of Serrana goats is characterized by sequential follicular wave growth with a great variability in their onset and duration, with the exception of the ovulatory wave. The temporal and morphological differences observed in the last wave of estrous cycle provide strong evidence for the role of progesterone in their regulation.

No abstract

The accuracy of transrectal real-time ultrasonography (RTU) scanning technique to detect ovarian structures (follicles and corpus luteum) of Serrana goats was compared to the data obtained by observation of ovarian sequential slices. This slicing technique (SLI) was considered as reference method. The laparoscopy and laparotomy techniques were also used for corpora lutea identification. For this purpose the ovaries of 14 females were observed, 7-8 days after ovulation, by transrectal ultrasonography followed by laparoscopic examination. Then ovaries were removed and studied by slicing. In the sliced sections of each ovary (n = 28), follicles and corpus luteum (CL) were identified and counted. CL and follicular diameters were measured using a millimetre scale.The total number of follicles, counted by RTU, was significantly lower than that observed by SLI (P < 0.01). This difference was mainly due to the under estimation of <2 mm follicles category. The correlation coefficient between category data obtained by RTU and SLI methods for the number of follicles ≥3 mm was high (r 2 = 0.95, P < 0.001), which highlights the use of UTR as a potential methodology to study the follicular dynamic of goats.There were no significant differences (P > 0.05) between the average number (mean ± S.D.) of corpus luteum identified per ovary by RTU (0.71 ± 0.75), laparoscopy (0.58 ± 0.71), laparotomy (0.67 ± 0.76) or SLI (0.83 ± 0.76) methods. The accuracy for the identification of ovulation, validated by CL detection on D7-D8 by SLI (100%), was 91.7%, 87.5% and 83.3% by RTU, laparotomy and laparoscopy, respectively. The negative predictive value of RTU, laparotomy and * Corresponding author. E-mail address: jsimoes@utad.pt (J. Simões). Simões et al. / Animal Reproduction Science 85 (2005) [263][264][265][266][267][268][269][270][271][272][273] laparoscopy to verify the absence of a CL in the ovary was 81.8%, 75.0% and 69.2%, respectively. The specificity of all three methods for the CL identification was 100%. No significant differences (P > 0.05) were found in the probability to detect the exact number of CL (0, 1 or 2) counted in each ovary between the RTU (87.5%), laparotomy (83.3%) and laparoscopy (75.0%) methods when compared with the reference method. 0378-The diameter of spherical CL could be estimated with reliability (r 2 = 0.86; P < 0.001). The real-time ultrasonographic scanning proved to be a highly accurate method for detection and measurement of several categories of follicles and CL size in Serrana goats. The results of the present study show that laparoscopy and RTU are similarly reliable techniques for CL detection. However, the RTU represents a non-traumatic technique with advantages to animal welfare both in experimental and reproductive evaluation of the size of ovarian structures.

AIMTo evaluate bacterial resistance to clarithromycin and fluoroquinolones in Brazil using molecular methods.METHODSThe primary antibiotic resistance rates of Helicobacter pylori (H. pylori) were determined from November 2012 to March 2015 in the Southern, South-Eastern, Northern, North-Eastern, and Central-Western regions of Brazil. Four hundred ninety H. pylori patients [66% female, mean age 43 years (range: 18-79)] who had never been previously treated for this infection were enrolled. All patients underwent gastroscopy with antrum and corpus biopsies and molecular testing using GenoType HelicoDR (Hain Life Science, Germany). This test was performed to detect the presence of H. pylori and to identify point mutations in the genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction from the biopsies, multiplex amplification, and reverse hybridization.RESULTSClarithromycin resistance was found in 83 (16.9%) patients, and fluoroquinolone resistance was found in 66 (13.5%) patients. There was no statistical difference in resistance to either clarithromycin or fluoroquinolones (P = 0.55 and P = 0.06, respectively) among the different regions of Brazil. Dual resistance to clarithromycin and fluoroquinolones was found in 4.3% (21/490) of patients. The A2147G mutation was present in 90.4% (75/83), A2146G in 16.9% (14/83) and A2146C in 3.6% (3/83) of clarithromycin-resistant patients. In 10.8% (9/83) of clarithromycin-resistant samples, more than 01 mutation in the 23S rRNA gene was noticed. In fluoroquinolone-resistant samples, 37.9% (25/66) showed mutations not specified by the GenoType HelicoDR test. D91N mutation was observed in 34.8% (23/66), D91G in 18.1% (12/66), N87K in 16.6% (11/66) and D91Y in 13.6% (9/66) of cases. Among fluoroquinolone-resistant samples, 37.9% (25/66) showed mutations not specified by the GenoType HelicoDR test.CONCLUSIONThe H. pylori clarithromycin resistance rate in Brazil is at the borderline (15%-20%) for applying the standard triple therapy. The fluoroquinolone resistance rate (13.5%) is equally concerning.