8resynchronization of Ovulation and Timed Insemination in Lactating Dairy Cows Using the Ovsynch and Heatsynch Protocols Initiated 7 Days Before Pregnancy Diagnosis on Day 30 by Ultrasonography

Abstract

We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-µG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4-8 × 106 mL −1 ) were incubated under non-capacitating conditions (modified glucose-free Tyrode's medium containing low bicarbonate, HEPES buffer, pyruvate and 3 mg mL −1 BSA V; 23 • C in air), and these spermatozoa remained alive for 24-48 h at 1G. To simulate µG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston,TX, USA) at 9 rpms. Spermatozoa were incubated in 1G and Sim-µG environments for 2.5-4.5 h and subsequently exposed to 0, 60 or 80 µg mL −1 LC for 0, 4, 8, 12, 16 and 20 min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status) + SYBR 14 (live status);(2) PI + FITC-PSA (acrosome reactions [ARs]); (3) PI + MitoTracker Deep Red (mitochondrial activity). Approximately 1 million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-µG + 60 µg mL −1 LC sperm were compared to 1G + 60 µg mL −1 LC, and 80 µg mL −1 LC sperm, their profiles were more similar to the 1G 80 µg mL −1 LC profiles. AR status: the Sim-µG + 60 µg mL −1 LC profiles were similar to the 1G + 60 µg mL −1 LC profiles. Mitochondrial Status: the Sim-µG + 60 µg mL −1 LC profiles were more similar to 1G + 80 µg mL −1 LC profiles. Summary: although Sim-µG sperm lost their motility within 3 h, they were alive. Cell profiles indicate that Sim-µG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-µG, induced changes, and whether Sim-µG sperm can penetrate eggs.

Effect of timing of initiation of resynchronization and presynchronization with gonadotropin-releasing hormone on fertility of resynchronized inseminations in lactating dairy cows

“…The strategy for resynchronization of ovulation on most dairy farms often relies on the timing of initiation of the Ovsynch protocol or variations of the Ovsynch protocol after insemination (Fricke et al, 2003;Bartolome et al, 2005;Sterry et al, 2006a). In most experiments, there are usually fewer pregnancies per AI (P/AI) to TAI after resynchronization compared with TAI at first service (Galvão et al, 2007;Silva et al, 2009;Thompson et al, 2010), indicating that fertility to TAI after resynchronization is not optimized.…”

Comparison of Oestrous Intensity between Natural Oestrus and Oestrus Induced with Ovsynch Based Treatments in Japanese Black Cows

“…2002). Yet, when distinct oestrous behaviour is not detected in Ovsynch based synchronization methods combined with timed artificial insemination (timed AI) protocol (Bartolome et al. 2005; Dalton et al.…”

“…The synchronization of oestrus or ovulation has been applied frequently to improve fertility and to achieve labour-saving at cattle farms (Pursley et al 1997;LeBlanc 2001;Gabor et al 2002). Yet, when distinct oestrous behaviour is not detected in Ovsynch based synchronization methods combined with timed artificial insemination (timed AI) protocol (Bartolome et al 2005;Dalton et al 2005), AI is not always performed by the AI operator.…”

Applications of Ultrasonography in Dairy Cattle Reproductive Management

“…The traditional defi nition of follicular cyst is a fl uid -fi lled, persistent structure greater than 2.5 mm. A more current defi nition proposed by Bartolome is the presence of multiple follicles greater than 18 mm, absence of a CL, and lack of uterine tonicity (Bartolome et al, 2005 ). Palpation has proven nearly useless for differentiating such structures from benign cysts (see #6 later), cavitary CLs, and soft CLs.…”