| 1. |
- Al-Makhzoomi, A., et al.
(author)
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Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden
- 2008
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 70:4, s. 682-691
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Journal article (peer-reviewed)abstract
- Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n = 8) and Swedish Holstein (SLB, n = 4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n = 3) or pooling two consecutive ejaculates (n = 104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing greater than 10% of morphologically deviating sperm head shapes, a commonly used threshold for young At bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires. (c) 2008 Elsevier Inc. All rights reserved.
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| 2. |
- Alminana, C, et al.
(author)
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Adjustments in IVF system for individual boars: Value of additives and time of sperm-oocyte co-incubation
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 64:8, s. 1783-1796
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Journal article (peer-reviewed)abstract
- In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5943 COCs were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa froth 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2 mM), hyaluronic acid (HA; [0.5 mg/mL]) and adenosine (10 mu M), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5 mg/ml) and adenosine (0, 10, 20 and 40 mu M) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 thin or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6 h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 1215 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P less than 0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9 +/- 3.9% versus 62.7 +/- 3.9% and 1.5 +/- 3.2 versus 1.3 +/- 3.5 for 10 min or 6 h, respectively), but reduced monospermy (P less than 0.001, 57.9 +/- 2.5% versus 70.0 +/- 2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF. (c) 2005 Elsevier Inc. All rights reserved.
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| 3. |
- Ballester, J., et al.
(author)
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Post-thaw viability of bull AI-doses with low-sperm numbers
- 2007
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 68:6, s. 934-943
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Journal article (peer-reviewed)abstract
- Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 106 total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/Pl), acrosome integrity (Carboxy-SNARF-1/PI/ FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols. (C) 2007 Elsevier Inc. All rights reserved.
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| 4. |
- Bolarin, A, et al.
(author)
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Dissimilarities in sows ovarian status at the insemination time could explain differences in fertility between farms when frozen-thawed semen is used
- 2006
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 65:3, s. 669-680
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Journal article (peer-reviewed)abstract
- Deep intrauterine insemination (DUI) offers a suitable alternative for the commercial use of frozen-thawed boar semen. The present study evaluated how the ovarian status at DUIs of frozen-thawed spermatozoa. (1 x 10(9) spz/dose, two DUls, 30-31 and 36-37 h after detection of oestrus) in 179 sows would explain differences in fertility between two farms with similar, but not equal, reproductive management (experiment 1). A further experiment investigated whether an increase in sperm number per AI-dose (1 versus 2 x 10(9) spz/dose, two DUls, 30-31 and 36-37 h after detection of oestrus, on 228 sows) could minimize this effect (experiment 2). Ovaries were checked by transrectal ultrasonography at the time of DUI and sows were classified into three categories: F-: ovarian pre-ovulatory follicles were visible during two examinations; O-: ovulation visible during one examination; and C-sows: corpora hemorragica visible during the two examinations. Overall farrowing rates differed (P less than 0.01) between farms (70.1 versus 51.2%, farms A and B, respectively). Distribution of sows among ultrasonography categories also differed (P less than 0.05) between farms (17.5, 72.2 and 10.3% were classified as F, O- and C-sows in farm A, versus 40.2, 29.3 and 30.5% in farm B). Nevertheless, farrowing rates and litter sizes within categories did not vary between farms (P greater than 0.05). In addition, a two-fold increase in the number of spermatozoa per DUI improved (P less than 0.05) fertility in F- and C-sows, but not in O-sows. In conclusion, the interval DUI-to-ovulation provides a major explanation for fertility differences between farms when frozen-thawed spermatozoa are used. (c) 2005 Elsevier Inc. All rights reserved.
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| 5. |
- Cox, J.F., et al.
(author)
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Computer-assisted analysis of sperm motion in goats and its relationship with sperm migration in cervical mucus
- 2006
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 66:4, s. 860-867
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Journal article (peer-reviewed)abstract
- In vitro sperm migration in cervical mucus relates to sperm concentration at the utero-tubal junction and to in vivo fertilization performance in goats. The present study aimed to characterize, using Computer-Assisted Sperm Analysis (CASA), motility patterns depicted by buck sperm and their relation to the migration efficiency in homologous (goat) and heterologous (heifer) cervical mucus in vitro. Semen was collected from 23 sexually mature bucks from three breeds by artificial vagina and sperm were assessed for motility parameters with a Hobson Sperm analyzer following extension in Sperm Analysis Medium (SAM). To study the relationship between kinematics parameters and the ability of sperm to migrate in cervical mucus, in a first experiment, motility performance of buck sperm suspended in SAM was compared against seminal plasma. In a second experiment, kinematics parameters of sperm were characterized. In a third experiment, bucks with sperm that differed in specific motion parameters were compared for the ability of their sperm to migrate through goat and bovine cervical mucus collected at estrus. In a fourth experiment, ejaculates that were compared in their migration ability and were assessed simultaneously for their motility parameters. Overall, sperm suspended in SAM medium had better velocity and similar linearity and lateral head displacement than those suspended in seminal plasma; furthermore, caprine sperm swam relatively fast (relative to bovine and ovine sperm), following a very linear trajectory. Under the conditions used, velocity parameters, linearity and lateral head displacement seemed to be related to sperm migration efficiency in homologous mucus but not in bovine cervical mucus. (c) 2006 Elsevier Inc. All rights reserved.
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| 6. |
- Cuello, C., et al.
(author)
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Vitrification of in vitro cultured porcine two-to-four cell embryos
- 2007
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 68:2, s. 258-264
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Journal article (peer-reviewed)abstract
- The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master (R) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n = 11) on day 2 (DO = onset of estrus). Some embryos (N = 63) were vitrified within 3 It after collection, warmed and cultured for 120 h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96 It in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24 h (Group VB; N = 65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N = 70) but were cultured in vitro for 120 h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6 +/- 0.7% and 3.2 +/- 0.5%, respectively). The survival and hatching rates of VB embryos (75.0 +/- 0.69% and 33.6 +/- 0.13%) were lower (p less than 0.001) than those obtained with control embryos (89.1 +/- 0.8% and 47.5 +/- 0.12%). Hatched VB embryos had a lower (p less than 0.01) total cell number than hatched control embryos (70.3 +/- 4.5 versus 90.6 +/- 3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master (R). (c) 2007 Elsevier Inc. All rights reserved.
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| 7. |
- Einarsson, S., et al.
(author)
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Conference Lecture: Influence of stress on estrus, gametes and early embryo development in the sow
- 2008
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 70:8, s. 1197-1201
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Journal article (peer-reviewed)abstract
- Systems with loose-housed sows have become common. Regrouping, which is commonly done after weaning and may coincide with many important reproductive events, causes stressful situations with elevated blood cortisol concentrations. Depending on group size, approximately 2-7 d are required for a new group of sows to become relatively stable. In a series of studies, the social stress after regrouping was simulated with repeated adrenocorticortriphic hormone (ACTH) treatments for approximately 48 h. Sows were allocated into control and experimental groups, fitted with jugular catheters, and blood samples were collected every 2 or 4 h. Follicular development and ovulation were monitored by transrectal ultrasonography every 4 h. Simulated stress during proestrus prolonged estrus and disturbed the follicular growth and ovulation. Giving ACTH during estrus elevated concentrations of cortisol and progesterone, and chagned the intraluminal environment, including exaggerated amounts of mucus in the UTJ and isthmus. Although ACTH had no effect on the time of ovluation (relative to onset of standing estrus), or on embryo development, fewer oocytes/embryos were retrieved from the ACTH group than from the control group (51% vs. 81%, P less than 0.05), and there was a tendency towards faster embryo transportation to the uterus. Short-term fasting after ovulation had an unfavourable effect on sperm numbers in UTJ/isthmus, cleavage rate of fertlized ova, as well as ova transport through the isthmic part of the oviduct. Treatment with ACTH after ovulation redcued numbers of spermatozoa at the zona pellucida and retarded cleavage rate of fertilized ova. Therefore, the timing of stress seemed to be an important factor regarding effects on reproductive events. (C) 2008 Elsevier Inc. All rights reserved.
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| 8. |
- Einarsson, S., et al.
(author)
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Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac (TM), on sexual maturity, reproductive organs and sperm morphology in male pigs
- 2009
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 71:2, s. 302-310
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Journal article (peer-reviewed)abstract
- The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac (TM) Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac (TM) vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n = 6) or 22 weeks (n = 6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination. and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p less than 0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p = 0.017-0.001). The immunization clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination. (c) 2009 Elsevier Inc. All rights reserved.
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| 9. |
- Ekwall, Hans, et al.
(author)
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Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks
- 2007
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 67:9, s. 1463-1472
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Journal article (peer-reviewed)abstract
- In this study we demonstrate, in the frozen state, the architecture of frozen boar spermatozoa collected from the sperm-rich fraction of ejaculates (n = 13) from four fertile boars packed and split-frozen in medium-straws (MS) and MiniFlatPacks (MFP), cross-sectioned in the frozen state and evaluated by image analysis on images obtained by use of cryo-scanning electron microscopy (Cryo-SEM). The tested hypothesis was that the degree of in situ dehydration and levels of homogeneity of boar semen either frozen in MSs or MFPs packages differ between them, with MFPs allowing for a more uniform dehydration of the spermatozoa and a higher cryosurvival, monitored by computer assisted sperm analysis (CASA) as proportion of linearly motile spermatozoa, compared to semen packaged and processed in MSs. The organization and relative surface of biological material (veins; e.g., frozen extender, bound water, solutes and spermatozoa) as well as free water (lakes) was measured as the degree of dehydration of the samples. The apparent organization of lakes and veins differed between packages, with the MFPs depicting larger lakes than the MSs. The sizes of the lakes in the latter appeared, moreover, highly asymmetrical depending on their position of the section. The relative surface of these lakes per section, respectively veins differed between packages (P less than 0.05), indicating a larger amount of free-water (lakes; 81.73 +/- 2.07% vs. 77.91 +/- 1.57%) in the MFPs and, consequently, thinner veins than in MSs. In conclusion, MFPs seem to allow for a more homogenous dehydration of the spermatozoa/frozen extender compared to MSs, which might account for their somewhat better sperm quality post-thaw. (C) 2007 Elsevier Inc. All rights reserved.
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| 10. |
- Gil, MA, et al.
(author)
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Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates?
- 2005
-
In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 64:2, s. 305-316
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Journal article (peer-reviewed)abstract
- The objective of this study was to determine if a multivariate pattern analysis of frozen-thawed sperm characteristics of boar semen of unknown fertility, thus identifying groups of ejaculates as "good" or "bad" freezers, would estimate their fertilizing potential in an in vitro embryo production (IVP) system. Frozen-thawed spermatozoa from a single ejaculate collected from 46 boars were evaluated for sperm motility and kinematic patterns, for sperm viability and for early changes in sperm membrane stability. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all ejaculates within a data set in to one of two groups, categorised as "good" (n = 25) or "bad" (n = 21) according with their freezability. In vitro matured oocytes were exposed to 2000 or 4000 frozen-thawed spermatozoa per oocyte for 6 h and then cultured in embryo culture medium for either 6 h (assurance of fertilization) or 7 days (to collect data on embryo development). Rates of sperm oocyte penetration and of embryo development significantly (p less than 0.05) increased in a sperm:oocyte ratio-dependent manner. A similar pattern was observed when sperm characteristics were grouped. Indeed, ejaculates classified as "good" showed significantly (p less than 0.05) higher rates of oocyte penetration, cleavage and of blastocyst formation than those classified as "bad". However, variation was still present among individuals (ejaculates, boars) in their ability to produce blastocysts in vitro. It is therefore concluded that despite the presence of a relationship for ejaculates with good semen quality post-thaw (thus grouped as "good") to higher IVP-results, the presence of individual variation does not allow for an accurate estimation of in vitro fertility based solely on the frozen-thawed semen quality parameters of a single ejaculate from a given boar. (c) 2004 Elsevier Inc. All rights reserved.
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| 11. |
- Hagman, Ragnvi, et al.
(author)
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Plasma PGF(2 alpha) metabolite levels in cats with uterine disease
- 2009
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In: Theriogenology. - : Elsevier BV. - 0093-691X .- 1879-3231. ; 72, s. 1180-1187
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Journal article (peer-reviewed)abstract
- Uterine disease induces PGF(2 alpha) increase in many animal species, which can be measured by the metabolite 15-keto-(13,14)-dihydro-PGF(2 alpha) (PGFM). Plasma PGFM levels are associated with severity of the uterine disease and presence of systemic inflammatory response syndrome (SIRS) in dogs. The objectives in this study were to investigate PGFM levels, presence of SIRS, and clinical and laboratory parameters in female cats as possible indicators for severity of uterine disease. In total, 7 female cats with pyometra, 2 with mucometra, 7 with cystic endometrial hyperplasia (CEH), and 14 healthy control cats were included. Physical examination, ovariohysterectomy, and histopathology were performed, laboratory parameters were analyzed, and PGFM levels were analyzed by radioimmunoassay. Analysis of variance, Fisher's exact test, Student's t-test and Pearson's product moment correlation coefficient were used for data analysis. In cats with pyometra, mean PGFM levels were increased (21.1 nmol L-1) but were decreased in cats with CEH (0.4 nmol L-1) compared with control cats (0.6 nmol L-1). In cats with mucometra, the mean PGFM level was 8.8 nmol L-1. Systemic inflammatory response syndrome was present in 6 (85%) cats with pyometra, 1 cat with mucometra, and 1 cat with CEH. Hospitalization length was negatively correlated with albumin and positively correlated with total white blood cell count (WBC), neutrophils, band neutrophils (BN), percentage BN (PBN), and monocytes. Pyometra and mucometra were associated with increased plasma levels of PGFM. The parameters albumin, WBC, neutrophils, BN, PBN, and monocytes may be useful to determine morbidity as measured by hospitalization length. (C) 2009 Elsevier Inc. All rights reserved.
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| 12. |
- Hallap, T, et al.
(author)
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Mitochondrial activity of frozen-thawed spermatozoa assessed by Mitotracker Deep Red 633
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 63:8, s. 2311-2322
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Journal article (peer-reviewed)abstract
- The present study was conducted to find a more objective method of evaluating sperm quality than the current subjective motility evaluations by testing the applicability of a novel fluorescent probe, Mitotracker Deep Red 633 (M-22426), for measuring the mitochondrial activity of spermatozoa both after freezing/thawing (PT) and after swim-up selection (SU), using flow cytometry (FC). The results from FC were compared to those of conventional microscopic motility evaluations and of computer-assisted sperm analysis (CASA) as well as to the fertility obtained after AI in the field. Semen from six Estonian Holstein bulls, processed when the sires were aged 3, 5, and 7 years, was included in the experiment. Sperm motility (measured either subjectively or by means of CASA) was always significantly (p less than 0.01-0.001) higher in the spermatozoa recovered by SU, for any of the age groups considered, or even when combining the age groups. Motility (measured subjectively) after SU was significantly (p less than 0.05) higher when bulls reached 7 years of age, compared to earlier collection ages, but no differences were registered between ages for CASA-assessed motility, either after SU or immediately PT. The numbers of spermatozoa. with high red fluorescence also increased after SU: p less than 0.05 (for semen from bulls aged 3 years), p less than 0.001 (5 years), p less than 0.001 (7 years), and p less than 0.001 when all age groups were combined. The proportion of spermatozoa with high mitochondrial activity as determined by Mitotracker Deep Red 633 correlated with sperm motility (p less than 0.01) both PT and after SU, but not with the fertility results. In conclusion, MitoTracker Deep Red 633 seems to be a reliable marker for frozen-thawed bovine semen viability both PT and after SU. (c) 2004 Elsevier Inc. All rights reserved.
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| 13. |
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| 14. |
- Hallap, T, et al.
(author)
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Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein Al bulls
- 2006
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 65:6, s. 1122-1136
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Journal article (peer-reviewed)abstract
- In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality Measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p less than 0.05), and membrane integrity (p less than 0.01). Following the SU selection, motility, membrane integrity (p less than 0.001). and membrane instability increased (p less than 0.01), as did stability (p less than 0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p less than 0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p less than 0.01), average path velocity (VAP) (p less than 0.001), and percentage of spermatozoa with unstable plasma lemma (p less than 0.05), had a significant relationship with non-return rate (NRR). The results indicate that it triple combination of the fluorophores M540/-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in it homogenous sire population.(c) 2005 Elsevier Inc. All rights reserved.
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| 15. |
- Hoflack, G., et al.
(author)
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Testicular dysfunction is responsible for low sperm quality in Belgian Blue bulls
- 2008
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 69:3, s. 323-332
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Journal article (peer-reviewed)abstract
- In a previous study, we demonstrated that Belgian Blue (BB) bulls have a higher prevalence of small scrota and poorer semen morphology compared to the Holstein Friesian (HF) breed in Belgium. The present study tested the hypothesis that the underlying reason for these BB traits negative to fertility was testicular degeneration, associated with an eventual hypoplastic background. At culling, sperm quality and testicular histology of BB bulls were assessed and compared to that of HF bulls. Besides semen quality being generally poorer in the BB breed, significantly more degenerative changes were encountered in BB compared to HF testicles (degeneration index: 37.7 +/- 11.9 versus 29.3 +/- 9.9 for BB and HF bulls, respectively; P = 0.053). These results correlated to the percentage of normal spermatozoa (r = -0.44; P = 0.024) and primary abnormalities (r = 0.38; P = 0.053). Moreover, the relative amount of collagen fibers present in the testicular interstitial connective tissue was correlated with % normal sperm (r = -0.47; P = 0.017), primary defects (0.48; P = 0.014), and the degeneration results (r = 0.63; P less than 0.001). The % testicular interstitial collagen fibers differed significantly between breeds (10.6 +/- 4.0% for the BB versus 7.6 +/- 1.9% for the HF bulls; P = 0.016). This increased amount of connective tissue in BB testes might hypothetically be responsible for the poorer sperm quality. This condition can be defined as a mild form of testicular hypoplasia, and might, in turn, be responsible for the higher sensitivity to testicular degeneration, which is encountered in the BB breed. (C) 2008 Elsevier Inc. All rights reserved.
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| 16. |
- Januskauskas, A, et al.
(author)
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Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 63:1, s. 160-178
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Journal article (peer-reviewed)abstract
- Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Triscitric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR- 14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P less than 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P less than 0.05), but had no significant effect (P greater than 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P greater than 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires. (c) 2004 Elsevier Inc. All rights reserved.
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| 17. |
- Kindahl, Hans
(author)
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15-Ketodihydro-PGF(2 alpha) and cortisol plasma concentrations in newborn foals after spontaneous or oxytocin-induced parturition
- 2009
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In: Theriogenology. - : Elsevier BV. - 0093-691X .- 1879-3231. ; 71, s. 768-774
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Journal article (peer-reviewed)abstract
- Hormonal changes during early neonatal life play a major role in the physiological processes underlying the maturation of several organs. Since prostaglandins and cortisol are associated with fetal organ system maturation, the aim of this study was to evaluate 15-ketodihydro-PGF(2 alpha) (PGM) and cortisol plasma concentrations during the first 21 days afterbirth in foals born by either spontaneous (24 foals) or low-dose oxytocin (OT)-induced parturition performed after at least 320 gestational days (25 foals) since induction is often considered to be a cause of prematurity. After spontaneous birth, the PGM concentration was significantly (P < 0.05) higher at 20 and 30 min compared to samples taken several hours or days later, while induced foals showed significantly (P < 0.05) higher concentrations at 10, 20, and 30 min. Regarding differences between the two groups, the plasma concentration of PGM was significantly higher 10 (P < 0.01), 20 (P < 0.05), and 30 (P < 0.05) min and 3 h (P < 0.05) after birth in induced foals compared to foals born by spontaneous parturition. It is difficult to determine whether the higher initial PGM concentrations in induced foals is related to higher uterine or fetal PGM release induced by exogenous OT stimulation. Cortisol plasma levels in both groups were higher at birth (P < 0.05) compared to the later sampling times. No differences were observed between the two groups indicating that the induction protocol used does not seem to result in premature foals. (c) 2009 Elsevier Inc. All rights reserved.
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| 18. |
- Kindahl, Hans
(author)
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Concentrations of 15-ketodihydro-PGF(2 alpha), cortisol, and progesterone in the plasma of healthy and pathologic newborn foals
- 2009
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In: Theriogenology. - : Elsevier BV. - 0093-691X .- 1879-3231. ; 72, s. 1032-1040
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Journal article (peer-reviewed)abstract
- Information regarding the plasma hormone profiles of prostaglandins (PGs), cortisol (C), and progesterone (P4) during pathologic processes in newborn foals is scarce. The aim of this study was to determine the plasma concentrations of these hormones in diseased foals (n = 40) and healthy at-term foals (n = 24) (Equus caballus) during the first 2 weeks of life. Blood samples were collected daily, before any treatment with nonsteroidal drugs in diseased foals, and plasma was analyzed by radioimmunoassay. 15-Ketodihydro-PGF(2 alpha) (PGM) was consistently higher in diseased foals than in healthy foals, probably related to roles of PGs in completing organ maturation and/or the presence of oxidative stress or inflammation. Similar trends were observed for C and P4. In diseased newborns, only PGM was significantly higher in nonsurviving foals, although C showed a similar profile. When specific diseases were considered, the levels of PGM and C were lower in premature foals at 12 h of life, whereas the concentration of P4 was higher than in controls. The results of this study demonstrate the differences in plasma hormone levels between healthy and pathologic newborn foals, particularly during the first 2 d of life, probably reflecting the inability of diseased foals to cope with the transition between fetal and neonatal life. (C) 2009 Elsevier Inc. All rights reserved.
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| 19. |
- Koonjaenak, S., et al.
(author)
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Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis)
- 2007
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 67:9, s. 1424-1435
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Journal article (peer-reviewed)abstract
- Altogether 218 frozen semen Al doses, prepared between 1980 and 1989 and also between 2003 and 2005 from 18 Al Thai swamp buffalo sires, were examined to determine whether seasonality affects post-thaw viability, as plasma membrane integrity (PMI, using SYBR-14/PI), plasma membrane stability (PMS, using Annexin-V/PI), or motility (Mot, using CASA). A thermoresistance test (38 degrees C for 60 min) was used to further analyze sperm survivability in vitro. All variables were compared over 3 seasons of the year (rainy: July-October; winter: November-February; and summer: March-June), with distinct ambient temperature and humidity. PMI (% of alive spermatozoa) was higher in winter (54.6%, P less than 0.001) than in the rainy (43.5%) or summer (46.7%) seasons. Outcomes of PMS (Annexin-V/PI assay) confirmed those of PMI, the highest PMS in spermatozoa processed in winter (55.7%, P less than 0.001). Spermatozoa depicting linear Mot post-thaw ranged from 48.2% to 48.8% across seasons (ns), proportions that decreased during incubation (33.5-37.9%), albeit without seasonal differences. The mean percentages of straight linear velocity (VSL), average path velocity (VAP), or curvilinear velocity (VCL) were higher (P less than 0.05-0.001) in the rainy season than in winter or summer, while average lateral head displacement (ALH) was higher (P less than 0.05) in summer, differences maintained after incubation. In conclusion, post-thaw PMS and PMI, assessed by flow cytometry, were significantly better in sperm samples processed during winter than in samples processed during the other seasons of the year, a seasonal difference not picked up by CASA, probably due to the larger number of spermatozoa assessed. (C) 2007 Elsevier Inc. All rights reserved.
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| 20. |
- Ljungvall, K, et al.
(author)
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Delayed effects on plasma concentration of testosterone and testicular morphology by intramuscular low-dose di(2-ethylhexyl)phthalate or oestradiol benzoate in the prepubertal boar
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 64:5, s. 1170-1184
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Journal article (peer-reviewed)abstract
- The immediate and delayed effects of prepubertal exposure to di(2-ethylhexyl)phthalate (DERP) or oestradiol benzoate on the plasma concentrations of testosterone, oestradiol and LH, as well as testicular morphology were examined in prepubertal boars. In a split litter design experiment, prepubertal boars were intramuscularly exposed to DEHP, oestradiol or vehicle during five weeks, starting at six weeks of age. The dose of DEHP was 50 mg/kg of bodyweight twice weekly, which is in the same range as recently used oral doses in rodents. Oestradiol-benzoate was administered at 0.25 mg/kg of bodyweight twice weekly. One set of animals was examined immediately after the exposure, and the other set was examined at an age of 7.5 months. During the exposure period concentrations of LH in plasma were lower (p = 0.02) in the oestradiol-treated animals than in the control group. In the group exposed to oestradiol, the relative to the body weight of the testicles tended to be lower (p = 0.07) than control immediately after five weeks of exposure, and the relative to the body weight of the seminal vesicles tended to be lower (p = 0.05) than control at 7.5 months of age. In the DEHP-exposed group an elevated (p = 0.005) concentration of testosterone and increased (p = 0.04) area of the Leydig cells in the testicles compared to the control group were seen at 7.5 months of age. These data suggest that DEHP early in life causes delayed effects on the reproductive system in the adult. (c) 2005 Elsevier Inc. All rights reserved.
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| 21. |
- Morrell, Jane, et al.
(author)
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Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily
- 2009
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 72:6, s. 879-884
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Journal article (peer-reviewed)abstract
- The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P less than 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 +/- 18.6%; SLC-Inc, 53.3 +/- 17.1%; SLC-Large, 44.9 +/- 18.3%) and were found to be equivalent in quality (motility: 88.0 +/- 8.8%, 84.0 +/- 3.5%, 90.0 +/- 5.4%; normal morphology: 69.4 +/- 17.0%, 69.4 +/- 12.7%, 63.9 +/- 15.6%; viability: 78 +/- 16.7%, 83.8 +/- 12.5%, 80.05 +/- 14.6%; DNA fragmentation index: 14.7 +/- 10.9%, 12.8 +/- 8.1%, 11.6 +/- 7.6%, respectively). The processing of an "average" stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use. (C) 2009 Elsevier Inc. All fights reserved.
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| 22. |
- Rodriguez-Martinez, Heriberto, et al.
(author)
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Boar spermatozoa in the oviduct
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 63:2, s. 514-535
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Journal article (peer-reviewed)abstract
- In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boars large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 23 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa. being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa - usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus - are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization. (C) 2004 Elsevier Inc. All rights reserved.
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| 23. |
- Rodriguez-Martinez, Heriberto, et al.
(author)
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Influence of seminal plasma on the kinematics of boar spermatozoa during freezing
- 2008
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 70:8, s. 1242-1250
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Journal article (peer-reviewed)abstract
- Sperm motolity is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozao susceptible to xodative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction of the ejaculate (portion 1. P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60 min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate poriton,s andapparently unrealted to changes in membrane integrity or membrane stability through conventiona, controlled cooling. (C) 2008 Elsevier Inc. All rights reserved.
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| 24. |
- Rodriguez-Martinez, Heriberto
(author)
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Role of the oviduct in sperm capacitation
- 2007
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 68, s. S138-S146
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Journal article (peer-reviewed)abstract
- Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation-capacitation in particular-is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization. (C) 2007 Elsevier Inc. All rights reserved.
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| 25. |
- Saravia, F, et al.
(author)
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Deep freezing of concentrated boar semen for intra-uterine insemination: effects on sperm viability
- 2005
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In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 63:5, s. 1320-1333
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Journal article (peer-reviewed)abstract
- The use of deep-frozen boar semen for artificial insemination (AI) is constrained by the need for high sperm numbers per dose, yielding few doses per ejaculate. With the advancement of new, intrauterine insemination strategies, there is an opportunity for freezing small volumes containing high sperm numbers, provided the spermatozoa properly sustain cryopreservation. The present study aimed to concentrate (2 x 10(9) spz/mL) and freeze boar spermatozoa packed in a 0.5 mL volume plastic medium straw (MS) or a multiple FlatPack (MFP) (four 0.7 mL volume segments of a single FlatPack [SFP]) intended as AI doses for intra-uterine AI. A single freezing protocol was used, with a conventional FlatPack (SFP, 5 x 10(9) spz/5 mL volume) as control. Sperm viability post-thaw was monitored as sperm motility (measured by computer-assisted sperm analysis, CASA), as plasma membrane integrity (PMI, assessed either by SYBR-14/PI, combined with flow cytometry, or a rapid hypo-osmotic swelling test [sHOST]). Sperm motility did not differ statistically (NS) between test-packages and control, neither in terms of overall sperm motility (range of means: 37-46%) nor sperm velocity. The percentages of linearly motile spermatozoa were, however, significantly higher in controls (SFP) than in the test packages. Spermatozoa frozen in the SFP (control) and MFP depicted the highest PMI (54 and 49%, respectively) compared to MS (38%, P less than 0.05) when assessed with flow cytometry. In absolute numbers, more viable spermatozoa post-thaw were present in the MFP dose than in the MS (P less than 0.05). Inter-boar variation was present, albeit only significant for MS (sperm motility) and SFP (PMI). In conclusion, the results indicate that boar spermatozoa can be successfully frozen when concentrated in a small volume. (C) 2004 Elsevier Inc. All rights reserved.
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