Classification of bovine reproductive cycle phases using ultrasound-detected features

With the combination of computer-assisted image analysis and ultrasonographic imaging technology, it has been possible to study and increase the knowledge in different areas of medicine. Studies of ovarian development in female mammals using ultrasonography have shown a relationship between the day in the estrous cycle and the main structures of the ovary.Ultrasound images of bovine ovaries were used to determine whether ultrasound-detected features can automatically determine the phase in the estrous cycle based on a single day's ultrasound examination of the ovaries. Five ultrasound-detected features of the bovine ovaries were used to determine the phase in the estrous cycle: (1) size of the dominant follicle; (2) size of the first subordinate follicle; (3) size of the second subordinate follicle; (4) size of the corpus luteum and (5) number of subordinate follicles with size ≥ 2mm. The collection of ultrasound images used for this study was formed by a group of 45 pairs of ovaries (left and right) which were imaged on day 3, day = 10 and day ≥ 17 of the estrous cycle corresponding to the metestrus, diestrus and proestrus phases respectively.Four different experiments were performed to test the hypothesis. For experiments 1, 2 and 3 the bovine ovaries were classified into three different classes: day 3 of wave 1 (D3W1), day 1 of wave 2 (D1W2) and day 17 or higher (D≥17) that were related to the follicular development of the ovary and the estrous cycle phases as: metestrus, diestrus and proestrus respectively. For experiment 4 the bovine ovaries were classified into four classes: D3W1, D6W1, D1W2 and D≥17. The additional class (D6W1: day 6 of wave 1) was incorporated to represent the early-diestrus phase in the estrous cycle.Two classifiers were implemented for all experiments and their performances compared: a decision tree classifier and a naive Bayes classifier. The decision tree classifier had the best performance with a classification rate of 100% for experiments 1, 2 and 3, giving a rather simple decision tree which used only two features to make a classification: size of the dominant follicle and size of the corpus luteum, suggesting these are key features in distinguishing between phases in the estrous cycle giving the most relevant information. The naive Bayes had a classification rate of 86.36% for experiment 1, 95.55% for experiment 2 and 90% for experiment 3. The results of this study supported the hypothesis that by using ultrasound detected features of bovine ovaries we can determine automatically the stage in the estrous cycle based on a single day's examination

Texture analysis of corpora lutea in ultrasonographic ovarian images using genetic programming and rotation invariant local binary patterns

Ultrasonography is widely used in medical diagnosis with the advantages of being low cost, non-invasive and capable of real time imaging. When interpreting ultrasonographic images of mammalian ovaries, the structures of interest are follicles, corpora lutea (CL) and stroma. This thesis presents an approach to perform CL texture analysis, including detection and segmentation, based on the classifiers trained by genetic programming (GP). The objective of CL detection is to determine whether there is a CL in the ovarian images, while the goal of segmentation is to localize the CL within the image. Genetic programming (GP) offers a solution through the evolution of computer programs by methods inspired by the mechanisms of natural selection. Herein, we use rotationally invariant local binary patterns (LBP) to encode the local texture features. These are used by the programs which are manipulated by GP to obtain highly fit CL classifiers. Grayscale standardization was performed on all images in our data set based on the reference grayscale in each image. CL classification programs were evolved by genetic programming and tested on ultrasonographic images of ovaries. On the bovine dataset, our CL detection algorithm is reliable and robust. The detection algorithm correctly determined the presence or absence of a CL in 93:3% of 60 test images. The segmentation algorithm achieved a mean (± standard deviation) sensitivity and specificity of 0:87 ± 0:14 and 0:91 ± 0:05, respectively, over the 30 CL images. Our CL segmentation algorithm is an improvement over the only previously published algorithm, since our method is fully automatic and does not require the placement of an initial contour. The success of these algorithms demonstrates that similar algorithms designed for analysis of in vivo human ovaries are likely viable

In Vitro Fertilization

The field of In Vitro Fertilization is a relatively new field in medicine, constantly on the move. This field is an exquisite example of the vast power in the complementary use of basic research with clinical practice and opened a new route of great basic and clinical research possibilities. The knowledge base that allowed the accomplishment of the idea of in vitro fertilization and embryo transfer has much developed since. The vast body of research pertaining to this field allowed deepening our understanding in the processes related to reproduction. In this book on in vitro fertilization we present new and interesting updated information in various aspects of this field. This work is a result of collaborative work of an international group of professionals dedicated to contribute to the advancement of our knowledge

Synchrotron imaging of bovine and human ovaries ex vivo

Background and Rationale: Reproductive dysfunction affects more than 15% of Canadian women; however, the underlying causes remain largely unknown. Ultrasonography is the most commonly used research and diagnostic tool for imaging the ovaries and uterus. However, current ultrasonographic techniques allow the detection of ovarian structures (eg. follicles, corpora lutea) at diameters of only ≥2 mm. The increased effectiveness of synchrotron technology for imaging ovaries in comparison to conventional imaging methods is currently unknown. Overall Objective: The overall objective of this research was to determine the effectiveness of synchrotron techniques for imaging ovaries. We hypothesized that synchrotron techniques would provide greater contrast for visualizing structural details of follicles, corpora lutea (CL), and cumulus oocyte complexes (COC), compared to conventional ultrasonography. Materials and Methods: Three studies were conducted to evaluate phase-contrast based synchrotron imaging methods. The first study involved Diffraction Enhanced Imaging (DEI) of bovine ovaries (n=6). The second study involved Propagation-Based Computed Tomography (PB-CT) imaging of bovine (n=4) and human ovaries (n=4). A third, preliminary study was conducted to explore the use of Talbot Grating Interferometry (TGI-CT) imaging of bovine (n=1) and human ovaries (n=1). Fresh and formalin-fixed bovine and human ovaries were imaged without or with contrast injection into the ovarian artery. Following synchrotron imaging, all ovarian samples were evaluated using diagnostic ultrasonography and histology. Images obtained using synchrotron techniques, ultrasonography and histology were qualitative and quantitatively compared. Results: DEI allowed the identification of 71% of follicles ≥2 mm and 67% of CL detected using ultrasonography. Mean follicle diameter was similar between DEI (9.6 ± 2.4 mm), ultrasonography (9.0 ± 2.6 mm), and histology (6.9 ± 1.9 mm) for fresh ovaries without contrast (P = 0.70). Likewise, no difference in CL diameter was detected between DEI (11.64 ± 1.67 mm), ultrasonography (9.34 ± 0.35 mm), and histology (9.6 ± 0.4 mm), (P = 0.34). Antral Follicle Count (AFC; ≥2mm) was similar between ultrasonography (6.5 ± 0.7 mm, fresh with no contrast; 6.5 ± 2.5 mm, preserved with no contrast) and DEI ( 4.5 ± 0.5 mm, fresh with no contrast; 6.5 ± 0.50 mm, preserved with no contrast) (P > 0.05). However, the contrast resolution for differentiating follicles and CL was inferior with DEI compared to ultrasonography. Small antral follicles <2mm, cell layers comprising the follicle wall and COC were not detected using either DEI or ultrasonography. PB-CT imaging enabled the visualization of 100% of follicles ≥2 mm and 100% of CL that were detected with ultrasonography. CL containing a central cystic cavity were identified using PB-CT; however, CL without a central cystic cavity were not well-visualized. Mean follicle and luteal diameters did not differ among PB-CT, ultrasonography and histology (P>0.05). PB-CT was superior to ultrasonography for detecting small antral follicles <2 mm in bovine ovaries (P = 0.04), and the granulosa and theca cell layers of the follicle wall in bovine and human ovaries (P < 0.0001). However, TGI-CT images exhibited greater contrast resolution for visualizing small and large antral follicles, CL, and the cell layers of the follicle wall compared to both PB-CT and ultrasonography. High contrast structures resembling COC were detected with both PB-CT and TGI-CT, but not with ultrasonography. Only TGI-CT permitted the visualization of the oocyte within the COC in fresh and preserved ovaries. Conclusions: DEI was inferior to ultrasonography for detecting ovarian follicles and CL. PB-CT was superior to ultrasonography for visualizing follicles <2 mm, COC, and the cell layers of the follicle wall. However, PB-CT was as effective as ultrasonography for detecting and measuring follicles ≥2 mm and cystic CL. Preliminary findings suggest that TGI-CT provides the greatest contrast for imaging both ovarian macro- and microanatomy compared to PB-CT, DEI, and ultrasonography

Actinomyces pyogenes in Embryonic Loss in Cattle

Actinomyces pyogenes is one of the bacteria commonly found in the bovine reproductive tract and it has been considered not to be a primary pathogen. The bacteria has also been isolated from foetuses and pus after abortion but its role as a primary or secondary pathogen in bovine abortion has remained an area of controversy and as yet little experimental work has been carried out to determine its role. In this thesis the potential role of A. pyogenes in bovine embryonic loss is examined

A bovine model to study reproductive aging

Decline in fertility with age has been well documented in women. There are ethical limitations to use humans as a model for basic research, and there is a lack of well characterized animal model. The objective was to characterize and validate a bovine model for the study of age-associated subfertility. All experiments were conducted on the same group of 13-14 year old cows (n=10), and their 1-4 year old young daughters (n=10). Mother-daughter pairs were used to reduce genetic variations. Follicular wave pattern in a natural reproductive cycle was maintained in old cows similar to that in daughters. We hypothesized that aging in cattle is associated with elevated circulating concentrations of FSH, and reduced concentrations of steroid hormones. As stated, circulating FSH concentrations were higher (P=0.009) during follicular waves in old than young cows. The ovulatory follicle in 2-wave cycles was smaller in old cows (P=0.04), but plasma estradiol concentrations were higher (P=0.01). Luteal phase progesterone tended to be lower in old than young cows (P=0.1). The number of 4-5 mm follicles recruited into a follicular wave was lower (

NEW FRONTIERS OF THE RESEARCH IN BUFFALO BREEDING

The interest around buffalo breeding in Italy has noticeably augmented during the last years, as demonstrated from the increase of buffalo population that passed from about 200,000 heads in 2000 to more than 400,000 in 2020. This is due to the interest around the main product derived from buffalo milk: the mozzarella cheese, also known as Mozzarella di Bufala Campana, that received in 1996 the Protected Designation of Origin (PDO). In Italy buffalo breeding is carried out in intensive and semi-intensive systems, similar to those utilized in dairy cattle: thus, similar issues in terms of productive and reproductive efficiency have been risen in this species. Furthermore, in the Mediterranean region, buffaloes show annual fluctuations in reproduction with distinct breeding and non-breeding seasons. As buffaloes are short-day breeders, the annual peak in fertility coincides with decreasing day length from autumn to winter. Therefore, in order to guarantee milk production throughout the year for mozzarella cheese production, some strategies are applied to increase the reproductive efficiency out of the breeding season, when a greater incidence of anestrus, a decline in the function of the corpus luteum (CL), and an increase in embryonic mortality are usually recorded. Furthermore, natural mating is often preferred to assisted reproduction (estrus synchronization and artificial insemination), because of some peculiarities of buffalo species, such as low estrus behavior and large variability in estrus duration, which makes difficult the individuation of the optimal moment for artificial insemination (AI). In this scenario, the purpose of this project was to improve reproductive efficiency and management in buffaloes through the application of new methodological approaches, such as synchronization of estrus, eco-color Doppler and metabolomics. In particular, a great attention has been paid to the improvement of AI in buffalo, in both heifers and pluriparous buffaloes. In the first experiment (Chapter 5), the efficiency of two synchronization protocols for oestrus synchronization and the influence of live body weight (LBW) and age on reproductive performance was evaluated in buffalo heifers. The animals were synchronized by Ovsynch-TAI Program (OVS; n=72) or double prostaglandin administered 12 days apart (PGF; n=74) and all the buffaloes were inseminated twice (24 days apart). Follicle dimensions and ovulation rate (OR) were assessed by ultrasound 24 and 48 h post-insemination. Pregnancy was assessed on day 25, 45 and 90 post-insemination and the incidence of late embryonic (LEM) and fetal (FM) mortality were respectively recorded. Data were analyzed by ANOVA, Chi-square test and multiple logistic regression. In the second experiment (Chapter 6), a deep study of CL development was carried out in buffaloes out of breeding season through the application of eco-color Doppler technique. Adult Mediterranean buffaloes (n=29) were synchronized by Ovsynch-TAI Program and artificially inseminated. CL B-mode/color Doppler ultrasonography examinations were performed daily from Days 5 to 10 post-synchronization, recording CL dimensions and blood flow parameters. Blood samples were collected on the same days for the progesterone (P4) assay. Data were grouped into pregnant or non-pregnant and retrospectively analyzed. In the third experiment (Chapter 7) a metabolomic approach on milk was used on 10 pregnant and 10 non-pregnant buffaloes in order to identify potential biomarkers of early pregnancy. The study was carried out on 10 pregnant and 10 non-pregnant buffaloes that were synchronized by Ovsynch-TAI Program and have undergone the first AI. Milk samples were individually collected ten days before AI (the start of the synchronization treatment), on the day of AI, day 7 and 18 after AI, and were analyzed by LC–MS. Data were analyzed retrospectively by dividing pregnant and non-pregnant subjects. Statistical analysis was carried out by using Mass Profile Professional. In the fourth experiment (Chapter 8) an advanced GC-MS and metabolite identification approach was also utilized to characterize the metabolome of buffalo milk and mozzarella cheese in a robust and repeatable technology platform. The study utilized eleven dairies located in a protected designation of origin (PDO) region and nine dairies located in non-PDO region in Italy. Samples of raw milk (100 mL) and mozzarella cheese (100 g) were obtained from each dairy and maintained at -80°C until analysis. Metabolomic assay was carried out through gas-chromatography and mass spectroscopy and differently expressed metabolites were identified. Statistical analysis of the results was carried out by ANOVA. The results of the first experiment showed that the LBW was significantly (P<0.05) higher in inseminated animals, compared to those that did not respond to the treatments (450.0±3.2 vs. 423.2±9.6 kg in inseminated and not inseminated heifers, respectively). Total OR was similar between groups, although OR at 24 h tended to be higher (P=0.06) in OVS (86.7 vs. 72.9% in OVS and PGF, respectively). A (P<0.01) higher LBW was observed in ovulated heifers of PGF, while no differences were recorded in OVS. LBW affected OR (odds ratio = 1,032; P < 0.05) only in PGF, while no effects were recorded in OVS. Total pregnancy rate, LEM and FM were similar between groups. In conclusion, the LBW would be considered before including buffalo heifers in a synchronization program and both synchronization treatments can be useful. In the second experiment, the total pregnancy rate was 50.0% (13/26) on Day 45. A significant difference between CL average area in pregnant and non-pregnant buffaloes was recorded only on Day 10. Pregnant buffaloes showed a significantly higher mean P4 concentration and higher mean time average medium velocity (TAMV) values from Day 5 to Day 10 compared to non-pregnant buffaloes. Linear regression analysis showed a significant relationship between P4 levels and TAMV. Multiple logistic regression highlighted a significant influence of TAMV on pregnancy outcome, particularly on Day 8. This is probably due to the strong relationship between TAMV and P4 production. Both TAMV and P4 could be used to predict pregnancy starting on Day 6, although a more reliable result was obtained at Day 10. Thus, the period between Days 5 and 10 is critical for CL development and pregnancy maintenance during the transitional period in buffalo. During the third experiment, Metabolomic analysis revealed the presence of several metabolites differentially expressed between pregnant and non-pregnant buffaloes. Among these, a total of five metabolites were identified by comparison with an online database and a standard compound as acetylcarnitine (3-Acetoxy-4-(trimethylammonio)butanoate), arginine-succinic acid hydrate, 5'-O-{[3-({4-[(3aminopropyl)amino]butyl}amino)propyl]carbamoyl}-2'-deoxyadenosine, N-(1-Hydroxy-2-hexadecanyl)pentadecanamide, and N-[2,3-Bis(dodecyloxy)propyl]-L-lysinamide). Interestingly, acetylcarnitine was dominant in milk samples collected from non-pregnant buffaloes. The results obtained from milk metabolic profile and hierarchical clustering analysis revealed significant differences between pregnant and non-pregnant buffaloes, as well as in the metabolite expression. Overall, the findings indicate the effectiveness of the metabolomic analysis for the identification of novel potential biomarkers in early prediction of pregnancy in buffaloes after AI, and these findings would give breeders the opportunity to rebreed animals at the next estrus event, saving most of the days as open. Finally, during the fourth experiment a total of 185 metabolites were consistently detected in both milk and mozzarella cheese. The PLS-DA score plots clearly differentiated PDO and non-PDO milk and mozzarella samples. For milk samples, it was possible to divide metabolites into two classes according to region: those with lower concentrations in PDO samples (galactopyranoside, hydroxybuthyric acid, allose, citric acid) and those with lower concentrations in non-PDO samples (talopyranose, pantothenic acid, mannobiose, etc.). The same was observed for mozzarella samples with the proportion of some metabolites (talopyranose, 2, 3-dihydroxypropyl icosanoate, etc.,) higher in PDO samples while others (tagatose, lactic acid dimer, ribitol, etc.,) higher in non-PDO samples. The findings establish the utility of GC-MS together with mass spectral libraries as a powerful technology platform to determine the authenticity, and create market protection, for "Mozzarella di Bufala Campana" PDO

Age-related changes in major ovarian follicle wave dynamics: morphologic and endocrinologic characteristics

Context: Reproductive aging results from depletion of the ovarian reserve and is marked by profound changes in hormone production and menstrual cyclicity. Age-related changes in antral follicle count and hormone production are well-documented. However, follicle wave dynamics and associated changes in hormone production have not been evaluated as women age. Overall Objective: To test the overall hypothesis that major follicle wave dynamics change as women age, and underlie changes in hormone production as women approach menopause. Materials and Methods: A prospective, observational study was conducted in 58 women of Reproductive Age (RA; 18-35 years; n=27), Advanced Reproductive Age-1 (ARA1; 36-44 years; n=10) and Advanced Reproductive Age-2 (ARA2; 45-55 years; n=21). The numbers and diameters of all follicles >2mm were recorded ultrasonographically every 2-3 days for 1 interovulatory interval. In a subset of women (RA, n=10; ARA2, n=17) blood samples were collected at each visit and serum was assayed for FSH, LH, estradiol, inhibin A and B, AMH and progesterone concentrations. Changes in AFC, follicle wave dynamics, and hormone production were compared between groups (SPSS v19.0, α=0.05). Results: The prevalence of Follicular and Luteal Phase Major Waves (FPMWs, LPMWs) was not different between RA, ARA1, and ARA2 groups [FPMW: 27/27(100%), 10/10(100%), 20/21(95%); LPMW: 10/17(37%), 3/10(30) %, 10/21(48%); P>0.050). All FPMWs were ovulatory. One LPMW ovulated at the time of menses in the ARA2 group; all other LPMWs were anovulatory. Dominant follicles in LPMWs emerged earlier (day -6, -2, -2; P=0.049), grew longer (11, 3, 6 days; P=0.005) and developed to a larger diameter (24, 11, 11 mm; P=0.032) in the ARA2 versus ARA1 and RA groups. In both RA and ARA2 groups, peak luteal phase estradiol concentrations were greater in women with (139.1, 177.8 pg/mL) versus without (78.0, 95.0 ng/mL) LPMWs (P=0.016, 0.074). In the RA group, 5/10 women developed LPMWs in association with higher mean (90.5 versus 53.3 pg/mL, P=0.028) and peak (139.1 versus 78.0 pg/mL, P=0.016) luteal phase estradiol concentrations versus women in the RA group without LPMWs, respectively. In the ARA2 group, LPMWs developed in association with atypical (>200ng/mL) elevations in estradiol concentrations in 4/8 women (50%). In all women (RA and ARA2 combined), the max diameter of the LPMW dominant follicle positively correlated with luteal phase estradiol (r=0.71, P6mm and inhibin B versus those without LPMWs. Polyovulatory FPMWs (n=3) were detected only in the ARA2 group and were associated with higher maximum follicular phase estradiol production and elevated luteal phase progesterone when polyovulation occurred at ovulation #1 of the IOI (n=2). Conclusions: The prevalence of FPMWs and LPMWs did not differ as women age. However, in women of advanced reproductive age, dominant follicles in LPMWs emerged earlier, grew longer and to a larger diameter. Approximately 50% of cases of LPMWs that developed in women of advanced reproductive age were associated with atypically high luteal phase estradiol and decreased progesterone. Women of reproductive age with a higher AFC >6mm and inhibin B were more likely to develop LPMWs

Ciste jajnika u goveda: pregledni članak

Ovarian cysts (OC) are one of the major factors affecting the fertility of dairy cattle due to their negative effects on reproductive performances, causing great economic losses. They have been traditionally defined as anovulatory follicular structures with a diameter exceeding 20 or 25 mm, lasting at least 10 days on the ovary in the absence of a functional corpus luteum. However, in recent years, the development of ovarian ultrasound and hormonal assays, particularly progesterone (P4), have provided additional information, thus changing the definition of this disease. These methods were proposed as the most effective combination for the diagnosis of OC. Treatments are primarily based on the use of different hormones or hormonal associations. Particularly, the standard treatment is GnRH. PGF2α is very effective in the case of luteal cyst. However, treatment failures observed with commonly administered hormones require the use of second-line treatments. Therefore, the Ovsynch protocol and progestogen appear to be a possible alternative in OC treatment. In order to prevent OC formation and to reduce OC incidence, strategies should focus on reducing diseases and stress and optimizing food intake during dry-off and post-partum (PP) periods. Medical prevention has been proposed with the same substances commonly used in treatment (GnRH, PGF2α).Ciste jajnika (OC) jedan su od glavnih čimbenika koji utječe na plodnost mliječne stoke uslijed njihovih negativnih učinka na reproduktivne pokozatelje, prouzročeći velike ekonomske gubike. Tradicionalno se definiraju kao anovulatorne folikularne strukture promjera većeg od 20-25 mm koje perzistiraju najmanje 10 dana na jajniku u izostanku funkcionalnog žutog tijela. Međutim, posljednjih godina, uporaba ultrazvuka u dijagnostici stanja jajnika, kao i hormonskih testova, posebice progesteronskih (P4), osigurao je dodatne informacije, mijenjajujući time definiciju ove bolesti. Ove metode predložene su kao najučinkovitija dijagnostička kombinacija za dijagnozu OCa. Glavne terapije baziraju se na uporabi različitih hormona ili njihovoj kombinaciji. Standardno liječenje posebice predstavlja uporaba GnRH. U slučaju luteinskih cista PGF2α je vrlo učinkovit. Međutim, neuspjesi liječenja zamijećeni s uobičajeno primijenjenim hormonima zahtijevaju drugu i drugačiju metodu liječenja. Stoga se Ovsynch protokol i gestageni čine kao moguća alternativa u liječenju OC-a. Kako bi se spriječilo formiranje OC-a i smanjila incidencija OC-a, strategije je potrebno usredotočiti na smanjenje bolesti i stresa i optimizaciju unosa hrane tijekom suhostaja i razdoblja nakon teljenja (PP). Predložena je medicinska prevencija s istim molekulama koje se uobičajeno rabe u liječenju(GnRH, PGF2α)

Molecular determinants of a competent bovine corpus luteum: first vs final wave dominant follicles

Reproductive management in cattle requires the synchrony of follicle development and oestrus before insemination. However, ovulation of follicles that have not undergone normal physiological maturation can lead to suboptimal luteal function. Here, we investigated the expression of a targeted set of 47 genes in (a) a first-wave vs final-wave dominant follicle (DF; the latter destined to ovulate spontaneously) and (b) 6-day-old corpora lutea (CLs) following either spontaneous ovulation or induced ovulation of a first-wave DF to ascertain their functional significance for competent CL development. Both the mass and progesterone-synthesising capacity of a CL formed following induced ovulation of a first-wave DF were impaired. These impaired CLs had reduced expression of steroidogenic enzymes (e.g. STAR and HSD3B1), luteotrophic receptors (LHCGR) and angiogenic regulators (e.g. VEGFA) and increased expression of BMP2 (linked to luteolysis). Relative to final-wave DFs, characteristic features of first-wave DFs included reduced oestradiol concentrations and a reduced oestradiol:progesterone ratio in the face of increased expression of key steroidogenic enzymes (i.e. CYP11A1, HSD3B1 and CYP19A1) in granulosa cells and reduced expression of the HDL receptor SCARB1 in thecal cells. Transcripts for further components of the TGF and IGF systems (e.g. INHA, INHBA, IGF2R and IGFBP2) varied between the first- and final-wave DFs. These results highlight the importance of hormones such as progesterone interacting with local components of both the TGF and IGF systems to affect the maturation of the ovulatory follicle and functional competency of the subsequent CL