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3.5th International Conference on Clinical & Experimental Cardiology, April 29, 2015, Philadelphia, USA
3
MORPHOLOGY OF THE GRASSCUTTER OVARIES: A TOOL FOR A BETTER HEART FUNCTION
Onoja, Benedict Onu
Department of Veterinary Anatomy, University of Nigeria, Nsukka
Nigeria
5.INTRODUCTION
Wild herbivorous rodent
( Hystricomorph )
Only in Africa (Baptist & Mensah,1986)
French-speaking African countries –
“agouti” (animal from the bush)-Adu et al.
(1999)
5
7. INTRODUCTION CONT’
Have short legs ( Adu, 2005)
Short tails with body covered with coarse,
bristly and even spiny hairs like porcupine
(Mensah & Okeyo, 2005)
Breeds all-year-round with peak incidence of pregnancy between June and July-months of greatest rainfall (Addo et al., 2007)
Narrow heads tapering rostrally (Ismail, 2008)
7
8. STATEMENT OF PROBLEM
Dependence on wild stock as source of grasscutter meat, has led to indiscriminate bush-burning, porching, destruction of forest reserves, environmental pollution & destruction of the ecosystem by depletion of the ozone layer.
8
9.STATEMENT OF PROBLEM CONT’
There are documented scientific reports on the:
nervous system (Byanet et al., 2008)
gastrointestinal tract (Byanet et al., 2008; Nwaogu and Adibe, 2010)
skeletal system (Byanet and Nwaogu, 2010)
respiratory system (Ajayi et al., 2009; Nwaogu and Aghaebita, 2010).
9
10.STATEMENT OF PROBLEM CONT’
Paucity of scientific information on the reproductive system of the doe, which could have facilitated its maintenance in captivity and help breeders expand their small scale to large scale necessitated this research.
10
11.JUSTIFICATION
Pilot farms by the Benin-German Government yielded favourable results in 1985 at Songhai Centre (Jori et al., 1995)
Source of protein and very low cholesterol
(NRC,1991)
Pets: Derivation of pleasure by the owners
11
12.JUSTIFICATION CONT’
Domestication: Grasscutter could be tamed &
used as laboratory animal (Addo, 1998)
Income: Grasscutters in captivity help
breeders expand from small scale to large
scale business (Adu & Yeboah, 2002)
Bomb Sniffing Research is On-going
12
13.RESEARCH QUESTIONS
What structural changes occur in the ovaries
of the female grasscutter as she attains
puberty?
Are the ovaries morphometrically
determined pre-natally?
At what age do female grasscutters attain
puberty?
Will the consumption of the grasscutter meat
better the heart function?
13
14.OBJECTIVES
MAIN OBJECTIVE:
To Evaluate Changes in the ovaries of the Grasscutter.
SPECIFIC OBJECTIVES:
To evaluate the morphometry of the ovaries
To evaluate the histomorphology of the ovaries.
14
15.MATERIALS & METHODS
Fifteen female grasscutters were purchased from David Mark Farms in Otukpo, Benue State.
Adminstration of multivitamin preparation were done prior to conveying the grasscutters (Adenkola et al., 2009)
Metallic cages as described by Raymond (2000) were used to convey the grasscutters to the experimental site
The grasscutters were divided into 3 groups of 5 each: Group A (1-3mths), Group B (4-6 mths) and Group C (7-9mths) respectively
15
16.MATERIALS & METHODS CONT’
GROSS:
Sedation-10mls of chloroform was applied unto a towel and put in a container.
Each grasscutter was anesthesized for 5 mins.
Live weight- A weighing scale with sensitivity of 20kg was used to weigh the grasscutters.
The grasscutters were ovarectomized.
16
17.MATERIALS & METHODS CONT’
Photography-Samsung digital camera (7.2 Megapixels)
was used to capture images in-situ.
Weighing of the ovaries using Mettler Balance with
sensitivity of 0.00001g.
Morphometry: Measurements of the ovaries vernier
calipers, thread and meter rule were done.
Standardized animal protocals were observed
throughout the research.
17
18.MATERIALS & METHODS CONT’
LIGHT MICROSCOPY:
Fixation- The ovaries were cut into blocks of 0.5cm in diameter & fixed by immersion in 10 % Neutral Buffered Formalin & 10 % Bouin’s fluid (300 mls of saturated aqueous solution of picric acid, 100 mls of formalin and 20 mls of glacial acetic acid as described by Drury et al., (1976) in a ratio of 1: 1 for 24hrs
Dehydration- The ovaries were passed through series of ascending ethanolic concentration of 70%, 80%, 90%, 95% and 100%
Clearing-The ovaries were passed through series of ascending concentrations of xylene (70%, 80%, 90%, 95% and 100%)
18
19.MATERIALS & METHODS CONT’
Embedding- The ovaries were embedded in molten paraffin wax (Junqueira, 2010)
Sectioning-Jung rotary microtome was used to section the embedded tissue to 6µm and placed in a floating-out bath maintained at 50ºC & mounted on a grease-free albumin-coated glass slide
Clearing- The tissues were dewaxed in series of descending concentrations of xylene (100%, 95%, 90%, 80%,70%)
19
20.MATERIALS & METHODS CONT’
Staining-The tissues were stained with
Haematoxylin and counter-stained with Eosin and
placed on a hot plate to dry
Microscopic Examination- The dried slides were
examined with Olympus microscope and appropriate
slides selected
Photomicroscopy-digital moticam MC 2001 1.3
Megapixels USB 3.0 was attached to a microscope
and images captured unto a laptop
20
21.DATA ANALYSIS
Statistical Package for Social Sciences
(SPSS) version 15 was used for data
analysis.
The data were analyzed using one-way ANOVA & Variant Means were separated using Duncan’s Multiple Range Test.
21
22.DATA ANALYSIS CONT’
Weights (g) & Lengths(cm) were expressed as Means ± Standard Errors of the Means.
Significance was accepted at p < 0.05.
22
23.23
FEMALE GRASSCUTTER
24.24
METALLIC GRASSCUTTER CAGE
25.25
GRASSCUTTER RESTRAINT
26.26
GRASSCUTTER FORAGE
27.27
GRASSCUTTER CONCENTRATE
28.28
RESULTS
& DISCUSSION
29.29
GIT
O
LEFT
RIGHT
Plate 1: Abdominal Cavity of the Grasscutter at 1-3 Months
showing the Ovaries (O), in-situ; Note that the
Gastrointestinal Tract (GIT) was laterally displaced to
expose the Ovaries.
Scale Bar =1cm.
30.Plate 2: Abdominal Cavity of the Grasscutter at 4-6 Months
showing the Ovaries (O), in-situ; Note that the
Gastrointestinal Tract (GIT) was laterally displaced to
expose the Ovaries. Scale Bar =1cm.
30
O
I
LEFT
GIT
RIGHT
31.
Plate 3: Abdominal Cavity of the Grasscutter at 7-9 Months
showing the Ovaries (O), in-situ; Note also that
the Gastrointestinal Tract (GIT) was laterally displaced to
expose the Ovaries. Scale Bar =1cm.
31
O
r
GIT
LEFT
RIGHT
32.32
TABLE 1: MEAN PERCENTAGE ORGANO-SOMATIC
INDICES (MPOSI) ± SEM OF THE LEFT
AND RIGHT OVARIES OF THE
GRASSCUTTERS
Different Superscripts abc in a Row Indicate Significant Difference at p < 0.05.
33.33
TABLE 2: MEAN RELATIVE LENGTHS (MRL) ± SEM
OF THE LEFT AND RIGHT OVARIES OF THE
GRASSCUTTER AND THEIR CROWN-RUMP
LENGTHS
Different Superscripts abc in a Row Indicate Significant Difference at p < 0.05.
34.34
Plate 7: Transverse Section of the Ovarian Cortex at 1-3 Months showing the Tunica Serosa (TS), Tunica Albuginea (TA), Pairs of Primordial Follicle (PF), Unilaminar Primary Follicle (UPF) and Primary Oocyte (PO). x400. H & E.
UPF
TA
PF
PF
TS
PO
50µm
PF
PF
50µm
Plate 8: Section of the Ovarian Cortex at 1-3 Months showing a “Nest” of Primordial
Follicles (PF). x400. H & E.
Plate 9: Transverse Section of the Ovarian Cortex at 4-6 Months showing the Ovarian Follicles at Different Stages of Development; Tunica Serosa (TS), Tunica Albuginea (TA), Primordial Follicle (PF), Unilaminar Primary Follicle (UPF) and Multilaminar Primary Follicle (MPF). x100. H & E.
MPF
UPF
TS
TA
50µm
PF
35.35
A
G
b
a
G
50µm
Plate 10: Section of the Ovarian Cortex at 7-9
Months showing two Secondary Follicles; (a)
Granulosa Cells (G) surrounding the Oocyte
without Antrum. (b) Granulosa Cells (G) with an
Antrum (A). x100. H & E.
Plate 11: Transverse Section of the Ovarian Cortex at 7-9 Months showing a Secondary
Follicle: Antrum (A), Centrally located Oocyte (O), Nucleus (N) of the Oocyte, Zona Pellucida (ZP), Multi-layered Granulosa Cells (G), and Tunica Albuginea (TA). Note the Theca Interna (TI) and Theca Externa (TE). x400. H & E.
TA
G
A
TI
TE
ZP
O
N
50µm
Plate 12: Ovarian Cortex at 7-9 Months showing Different Stages of Follicular Development; Primordial Follicle (PF), Unilaminar Primary Follicle (UPF) and Graafian Follicle (GF); (a) Antrum (A), Corona Radiata (CR), Cumulus Oophorus (CO), Zona Pellucida (ZP), Granulosa Cells (G), Oocyte (O) at the centre of the ovary. (b) Oocyte (O) at the periphery of the ovary. x100. H & E.
A
ZP
CO
G
UPF
PF
O
a
b
GF
50µm
CR
CR
CR
O
36.36
CH
I
50µm
Plate 13: Section of the Ovarian Cortex at 7-9 Months showing the Corpus Haemorrhagicum (CH) indicated by blood clot (Arrow) within the inner wall of the Graafian Follicle. Note the Indentation (I) on the Ovarian wall. x400. H & E.
Plate 14: Transverse Section of the Ovarian Cortex at 7-9 Months showing the Corpus Luteum (CL), Remnants of Granulosa Cells (G), Remnants of Cumulus Oophorus (CO) and Liquor Folliculi
(Arrow). x100. H & E.
Plate 15: Section of the Ovarian Cortex at 7-9 Months showing Developing Follicles (D),
Degenerating Follicle (DF) and Atretic Follicle (AF). x100. H & E.
CL
CO
G
50µm
DF
AF
D
50µm
37.SUMMARY
GROSS
The ovaries were spindle-shaped at 1-3
moths of age.
The ovaries were oval at 4-6 months of age.
The ovaries were round at 7-9 months of
age.
37
38.SUMMARY CONT’
MORPHOMETRY
The MPOSI & MRL of the Ovaries Increased
Significantly as the Animal Attained Puberty.
The Left Side was Significantly Higher
than the Right in All the Age Groups.
The Weights & Lengths of the Ovaries were
not Determined Pre-Natally.
38
39.SUMMARY CONT’
HISTOMORPHOLOGY
Centrally Located Oocyte in the Ovarian Follicle Both
at 1-3 & 4-6 Months of Age.
Peripherally located Ooctye in the Ovarian Follicle at 7-9
Months of Age.
Corpora Haemorrhagica & Lutea that were absent @ 1-3
& 4-6 Months of Age in the Ovarian Follicle, were
Present @ 7-9 Months of Age.
39
40.CONCLUSION
Puberty in the Female Grasscutters was
Attained @ 7 Months of Age.
Breeders & Reseachers can Avail
themselves of this Information; so as to
know when to Breed their Female
Grasscutters.
40
41.CONCLUTION CONT’
Grasscutter meat, being white, has
very high protein content but low
cholesterol level (NRC,1991).
Thus, consumption could reduce
cardiovascular disorders like
arteriosclerosis, high blood pressure,
congestive heart failure, stroke and
even death.
41
42.CONTRIBUTION TO KNOWLEDGE
This is the First Time a Concise Study
of the Gross, Morphometry &
Histomorphology of the Ovaries of the
Grasscutter @ the Light Microscopic
Level would be Carried Out.
42
43.RECOMMENDATION
More Research on the Female
Reproductive System of the
Grasscutter Using Electron
Microscopic Technique is
Advocated so as to Elucidate
Minute Details.
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44.44
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45
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